Preparation of Fucoxanthin-Loaded Nanoparticles Composed of

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Preparation of Fucoxanthin-Loaded Nanoparticles Composed of Casein and Chitosan with Improved Fucoxanthin Bioavailability Song Yi Koo, Il-Kyoon Mok, Cheol-Ho Pan, and SANG MIN KIM J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04376 • Publication Date (Web): 23 Nov 2016 Downloaded from http://pubs.acs.org on December 6, 2016

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Journal of Agricultural and Food Chemistry

Preparation of Fucoxanthin-Loaded Nanoparticles Composed of Casein and Chitosan with Improved Fucoxanthin Bioavailability

Song Yi Koo†, Il-Kyoon Mok†,§, Cheol-Ho Pan†, Sang Min Kim†,*

†

Systems Biotechnology Research Center, KIST Gangneung Institute of Natural Products,

Gangneung, Gangwon-do 25451, Republic of Korea

§

Department of Food Processing and Distribution, Gangneung-Wonju National University,

Gangneung, Gangwon-do 210-702, Republic of Korea

*Convergence Research Center for Smart Farm Solution, KIST Gangneung Institute of Natural Products, Gangneung, Gangwon-do 25451, Republic of Korea

*Corresponding author: Sang Min Kim, Tel: +82-33-650-3640, Fax: +82-33-650-3679 E-mail: [email protected]

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ABSTRACT

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To facilitate the utilization of fucoxanthin (FX), a valuable marine carotenoid, in the food

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industry, FX-loaded casein nanoparticles (FX-CN) and chitosan-coated FX-CN (FX-CS-CN)

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were developed using the FX-enriched fraction from Phaeodactylum tricornutum. Two

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nanoscale particles (237 ± 13 nm for FX-CN and 277 ± 26 nm for FX-CN-CN) with spherical

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and smooth surface showed over 71% encapsulation efficiency and polydispersity index (PDI)

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value of 0.31~0.39 in water. Owing to the chitosan coating, FX-CS-CN showed a positive

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zeta potential (24.00 mV), whereas that of FX-CN was negative (−12.87 mV). In vitro-

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simulated digestion demonstrated better FX bioaccessibility from the nanoparticles versus P.

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tricornutum powder (Pt-powder), and from FX-CN versus FX-CS-CN. However, in C57BL/6

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mice, fucoxanthinol absorption to the blood circulation was two times higher for FX-CS-CN

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versus FX-CN, possibly due to increased retention or adsorption to mucin by the cationic

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biopolymer in the chitosan-coated particles. These results demonstrate that FX-CS-CN can

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enable the application of FX, with improved bioavailability and water dispersibility, in the

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food industry.

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KEYWORDS: bioavailability; casein; chitosan; fucoxanthin; nanoparticles; Phaeodactylum

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tricornutum

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INTRODUCTION

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Fucoxanthin (FX), a marine xanthophyll carotenoid, is abundantly found in macroalgae such

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as Laminaria japonica, Undaria pinnatifida, and Ecklonia cava.1 Until now, multiple health-

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promoting properties, such as antioxidant, anticancer, and anti-inflammatory activities, have

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been reported for FX.1 Among these properties, the antiobesity activity is the most promising

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effect, and this has been supported by multiple in vitro and in vivo studies.1 Recently, some

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microalgae, including Phaeodactylum tricornutum, Odontella aurita, and Isochrysis galbana,

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were suggested as new sources for FX production.2-4 However, the global production and

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distribution of FX from macro- or microalgae is very limited compared to those of lutein and

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astaxanthin, which are two popular carotenoids from microalgae, owing to the difficulty of

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achieving cost-effectiveness for the biomass production and extraction of FX. Most FX-

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related products advertising an antiobesity effect, which are currently available on the market,

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contain simple ethanolic extracts from macroalgae as the FX source. However, simple algal

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extracts are not suitable for application in the food industry owing to their unique odor, sticky

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consistency, and low concentration of FX. Thus, improved purification and processing

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techniques are required to facilitate the wider utilization of FX in the food industry and other

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fields.

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Like other carotenoids, FX is sensitive to light, oxygen, and pH.5 To seek a solution for this

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problem of low stability, several emulsion and nanoparticle techniques have been evaluated

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and FX has been demonstrated to have increased stability and bioavailability inside these new

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materials. Liquid systems comprising FX emulsions from micro to nano scales have been

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produced based on triglycerols and canola oil, and these have been shown to reduce the

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degradation of FX by heat, oxygen, and light.5,6 The bioaccessibility of FX under in vitro

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digestion and animal feeding studies was also found to be improved to different extents 3 ACS Paragon Plus Environment


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according to the type of carrier oil used in the following order : long chain triacylglycerols ˃

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medium chain triacylglycerols ˃ indigestible orange/mineral oils.7 A solid system composed

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of chitosan, sodium tripolyphosphate, and glycolipid was used for FX encapsulation by an

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ionic gelation method, whereby FX demonstrated increased stability and bioavailability.8

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Sodium caseinate, produced by neutralization and spray-drying of the precipitated caseins

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from skimmed milk, is commonly used as an emulsifying, stabilizing, and delivering agent in

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the food and pharmaceutical industries.9 It has excellent surface activity and shows unique

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self-assembly, as well as abundance and low cost.10 Four major components of caseins (αs1-,

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αs2-, β-, and κ-casein) show amphiphilic properties with high proportions of hydrophobic and

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hydrophilic amino acid residues.11 Therefore, casein can self-assemble easily to generate

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stable spherical micelles with an average diameter of 150 nm and form complexes with

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bioactive compounds to improve their stability and bioavailability. Another beneficial aspect

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of sodium caseinate is the ease of preparing it as a powdered form by drying techniques such

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as spray-dying and freeze-drying for the removal of solvents and convenience of

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transportation, storage, and application in the food and pharmaceutical industries.12 Bioactive

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compounds with a low aqueous solubility, stability, and bioavailability, such as flutamide (an

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anti-androgenic agent), bixin (the major colorant component of annatto), curcumin (a natural

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polyphenol from the rhizome of turmeric), and β-carotene (a carotenoid precursor of vitamin

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A) have been used to make mixed micelles with sodium caseinate, and their properties were

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characterized with respect to the stability and bioavailability of bioactive compounds.10-13

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Meanwhile, chitosan is a cationic polysaccharide under neutral or basic pH conditions and

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can be obtained by deacetylation of chitin which is the most widely distributed biopolymer in

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nature and produced industrially from the crustacean wastes. By controlling the degree of

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deacetylation and molecular weight of chitosan, this biopolymer can be useful as an

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encapsulation material for micro/nanoparticles. The main advantages of using chitosan in 4 ACS Paragon Plus Environment

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micro/nanoparticles include the controlled release of active agents, cross-linking ability,

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cationic nature for a mucoadhesive character, and solubility in aqueous acidic conditions.14 A

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number of methods have been developed to prepare micro/nanoparticles of chitosan. These

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include emulsion cross-linking, coacervation/precipitation, spray-drying, emulsion-droplet

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coalescence, ionic gelation, reverse micellar extraction, and sieving methods.6, 14

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The purpose of this study was to develop and characterize FX-loaded nanoparticles with

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casein (FX-CN) and FX-CN coated with chitosan (FX-CS-CN) from the microalga P.

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tricornutum, to facilitate the wider utilization of FX in the food industry. The characteristics

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of the nanoparticles were investigated, including their size, morphology, structure, zeta

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potential, polydispersity index (PDI), encapsulation efficiency, and adsorption to mucin. In

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addition, the release of FX from the matrix in the gastrointestinal (GI) tract and the

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absorption of fucoxanthinol (FXOH), a primary metabolite of FX by lipase (Figure 1A), into

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the mouse blood circulation system were tested, with P. tricornutum dry powder as a

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comparator, by an in vitro simulated digestion assay and an in vivo pharmacokinetic study,

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respectively, to evaluate the bioaccessibility and bioavailability of FX from FX-CN and FX-

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CS-CN.

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Materials and Methods

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Materials. The powder of P. tricornutum (Pt-powder) and an FX-enriched fraction (FX-fr)

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containing over 30% (w/w) of FX (as shown in Figure 1B) were provided from BioOne Co.

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Ltd. (Gangneung-si, Gangwon-do, Korea). FX-fr was produced by partial purification of FX

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from the ethanolic extract of Pt-powder through the silica gel chromatography method

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modified from our previous report. 2 Casein sodium salt from bovine milk, chitosan (medium

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molecular weight), Tween® 80, xanthan gum, glycerol, and calcium chloride were purchased 5 ACS Paragon Plus Environment


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from Sigma-Aldrich (St. Louis, MO, USA). All solvents were of HPLC grade and were

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purchased from Sigma-Aldrich and Fisher Scientific Korea Ltd (Seoul, Korea).

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Preparation of FX-CN and FX-CS-CN. Two types of FX-loaded nanoparticles were

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prepared based on the method described by Pan et al.12 Three grams of casein sodium salt

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was dissolved in 150 mL of 40% (v/v) aqueous ethanol by moderate stirring with slight

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heating for 120 min. After the sodium casein became totally hydrated, an ethanolic solution

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(2 mL) of 12 mg mL-1 FX-fr was added to 150 mL of 2% sodium caseinate solution while

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stirring for 60 min. Then, 35 mL of 0.1 M K2HPO4 and 3 mL of 0.4 M tripotassium citrate

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were added, followed by a dropwise addition of 110 mL 0.03 M CaCl2 and blending at 15000

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rpm for 5 min using a high speed homogenizer (ULTRA-TURRAX® T18, IKA, Stauffen,

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Germany). After homogenization, the mixture was injected into the electrospray system,

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using a syringe pump. Then, the mixture solution was sprayed through the nozzle while

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applying a fixed voltage of 5 kV and a flow rate of 130 µL/min to obtain FX-CN. FX-CS-CN

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was prepared by a chitosan coating process with the FX-CN solution. Chitosan solution

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(0.1%, w/v) was prepared by dissolving chitosan in distilled water containing 0.1% (w/v)

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acetic acid. To remove the impurities, chitosan solution was filtered using a 0.45-µm syringe

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filter (Minisart®, Sartorius AG, Göttingen, Germany). The prepared chitosan solution was

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added dropwise to FX-CN solution and stirred mildly for 1 h to obtain FX-CS-CN. Two

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freshly prepared nanoparticle dispersions were used for the measurement of various

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characteristics and then the nanoparticles were freeze-dried at −120 ℃ for 48 h using a

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freeze-drier (FDCF-12003, OPERON, Kimpo, Korea), and the dried samples were stored in

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glass vials at −20 ℃ until use.

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Characterization of FX-CN and FX-CS-CN. The mean particle size, PDI, and zeta

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potential of FX-CS-CN and FX-CN were measured using dynamic light scattering with a

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NanoZS (Malvern Instruments, Worcestershire, UK) at 25 ℃, with a detector angle of 90°A

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minimum of three parallel measurements were performed and the average value was

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calculated.

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Structural characterization of the two types of nanoparticles was performed by Fourier

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transform infrared (FT-IR), using a model V430 apparatus (Jasco, Tokyo, Japan) at 20 ℃. The

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FT-IR spectra of FX-fr, chitosan, casein, FX-CN, and FX-CS-CN were obtained to estimate

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the interactions between chitosan and casein. All samples were mixed with potassium

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bromide (KBr) at a ratio of 1:10 and formed into pellets by compression under a force of 5

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tons in a hydraulic press. These pellets were scanned in transmission mode. Each spectrum

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was obtained from the average of 64 scans at a resolution of 4 cm-1 in the wavelength range

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of 600−4000 cm-1.

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The morphologies of FX-CN and FS-CS-CN were examined by field emission scanning

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electron microscopy (FE-SEM). To prepare SEM samples, the samples were mounted on FE-

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SEM stubs with double-sided adhesive tape and coated under vacuum with platinum to make

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the samples conductive. Samples were observed using an FE-SEM instrument (SU70, Hitachi,

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Tokyo, Japan) at an accelerating voltage of 15 kV. The photographs were taken at selected

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magnifications and representative images were reported.

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To evaluate FX stability in the nanoparticles, two nanoparticles were dissolved in water

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and adjusted to the final FX concentration of 8 µg/mL. After then, these aqueous solutions

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were stored at three different temperatures (2 ℃, 10 ℃, 26 ℃) for 4 weeks under darkness. FX

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contents were analyzed every week by HPLC.

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Accurately weighed amounts (0.1 g) of FX-CS-CN and FX-CN were dispersed and

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dissolved in ethanol and sonicated for 1 h. The encapsulation efficiency of the nanoparticles 7 ACS Paragon Plus Environment


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was determined using the equation below:

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Encapsulation efficiency (%) = Fa/Fth × 100

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where Fa is the actual amount of FX encapsulated in FX-CS-CN and FX-CN and Fth is the

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theoretical amount, assuming that all of the FX added in this experiment was encapsulated

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within the nanoparticles without any losses during the preparation procedure.

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A mucin adsorption test was performed using a slight modification of a previously

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reported method by Filipović-Grčić et al. 15 and the amount of free mucin was calculated by

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the Bradford method.16 Mucin solution (1 mL of 1 mg/mL concentration) was blended with 1

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mL of FX-CN or FX-CS-CN at 37 ℃ for 1 h. Then, the suspension was centrifuged at 15000

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rpm at 4 ℃ for 30 min to isolate the free mucin (supernatant) and adsorbed mucin (pellet). In

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the Bradford method, Bradford reagent was diluted 5-fold and mixed with the supernatant.

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Then, the mixture was incubated in a 37 ℃ water bath with shaking at 180 rpm for 10 min.

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The quantity of mucin in the supernatant was calculated by measuring the absorbance at 595

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nm. To make the standard curve, mucin solutions of 31.25, 62.5, 125, 250, 500, 1000, and

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2000 µg/mL were prepared and the absorbance of each solution was measured. Mucin

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adsorption (%) was calculated using the equation below:

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Mucin adsorption (%) = [(Ct-Cf)/Ct] × 100 where Ct is the total amount of mucin and Cf is the free mucin in the supernatant.

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In vitro simulated digestion. Simulated digestion was performed according to the method

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described by Garrett et al.,17 with slight modifications. To test in vitro simulated digestion,

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digestive steps were performed sequentially. Briefly, FX-CS-CN or FX-CN powder (25 mg)

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was homogenized in 10 mL saline solution containing 120 mM NaCl, 5 mM KCl, and 6 mM

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CaCl2 (pH 5.5). Then, 1000 units of α-amylase was added, and the pH was adjusted to 6.5.

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After the saline solution had been added to a volume of 12.5 mL, the samples were incubated 8 ACS Paragon Plus Environment

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at 37 ℃ for 5 min in a shaking water bath (Lab Companion, Jeio Tech, Seoul, Korea) at 95

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rpm to simulate the oral phase of digestion. To mimic the gastric phase of human digestion,

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the pH of the sample was acidified to 2.2 with HCl and 0.5 mL of a porcine pepsin solution

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(0.075 g/mL in 0.1 N HCl) was added. The samples were suspended in a saline solution to a

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volume of 15 mL and incubated at 37 ℃ for 2 h. Three intestinal parts (duodenum, jejunum,

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and ileum) were separated and simulated sequentially. To simulate the duodenum stage, 250

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mg of bile extract, 0.5 mL of pancreatic lipase (0.01 g/mL), and 0.5 mL of pancreatin (0.08

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g/mL) were added and the pH was increased to 5.5 by adding 1 M sodium bicarbonate. The

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samples were incubated for 30 min at 37 ℃ (final volume of 20 mL). To mimic the jejunum

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stage, the pH was adjusted to 6.0 and the samples were incubated for 90 min at 37 ℃ (final

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volume of 22.5 mL). Finally, the pH was adjusted to 7.0 and the samples were incubated for 5

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h at 37 ℃ (final volume of 25 mL) to simulate the ileum stage of digestion.

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Pharmacokinetic study. In vivo animal study was adhered to the Guide for the Care and Use

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of Laboratory Animals developed by the Institute of Laboratory Animal Resources of the

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National Research Council. The protocol for the pharmacokinetic study was approved by the

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Institutional Animal Care and Use Committee of KIST (2015-018) in Seoul, Korea. For a

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single dose administration corresponding to 7 mg FX/kg mouse body weight, the mice (male

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C57BL/6, 7 weeks old, 25~50 g) were administered with the powder type samples (Pt-

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powder, FX-CN, and FX-CS-CN) dissolved in distilled water by intragastric gavage. The

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animals were sacrificed at 0.5, 1, 2, 4, 8, and 24 h after administration. The control mice at

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the 0 h time point were not orally administered anything, but just anaesthetized. The blood

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was collected by cardiac puncture in a heparinized syringe and was centrifuged at 1,000 × g

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for 10 min at 4 ℃. The supernatant fraction was used as the plasma samples. The plasma 9 ACS Paragon Plus Environment


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concentrations of FXOH were determined by extracting FXOH from plasma samples with

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ethyl acetate solvent and performing liquid chromatography/tandem mass spectrometry (LC-

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MS/MS) analysis. As an internal standard, carbamazepine was used. The pharmacokinetic

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study of each treatment group was measured with three mice (n = 3). Non-compartmental

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pharmacokinetic parameters of FXOH were calculated from the time course of plasma

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concentration by WinNonlin® program (Pharsight, Mountain View, CA, USA).

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High pressure liquid chromatography and LC-MS/MS conditions. For the in vitro

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simulated digestion assay, the target compounds (FX and FXOH) were quantified using a

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high pressure liquid chromatography system coupled to a diode array detector (1200 series,

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Agilent, Santa Clara, CA, USA). A YMC C-30 carotenoid column (150 × 4.6 mm internal

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diameter, 3 µm particle size, Waters, Milford, MA, USA) was used for the separation. A

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methanol and water solvent system was used as the mobile phase at a flow rate of 0.7 mL/min

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with a column temperature of 35 ℃. The solvent gradient program was as follows: the

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methanol/water ratio was increased from 90:10 to 100:0 over 20 min, and then held at 100%

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methanol for the next 5 min. The chromatogram obtained at 450 nm was used for the

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quantitative analysis of FX and FXOH. For the pharmacokinetic study, FXOH in mouse

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plasma samples was identified and quantified using an LC-MS/MS system (1200 series

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instrument coupled with a 6410 series instrument, Agilent). Multiple reaction monitoring

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modes were used for the quantification: m/z 617.1 > 109.2 for FXOH and m/z 237 > 194 for

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carbamazepine (used as a standard reagent).

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Statistical analysis. All experiments were repeated in triplicate. All data are presented as

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mean ± standard deviation. The significant differences among means were analyzed by t-test,

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one-way ANOVA (P < 0.05) and Duncan’s multiple range test (DRT). 10 ACS Paragon Plus Environment

The t-test and one-

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way ANOVA analysis were performed by Microsoft Office Professional Plus 2010 Excel and

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DRT was conducted by the free statistical software R program (version 3.3.2).

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RESULTS AND DISCUSSION

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Characterization of FX-CN and FX-CS-CN. The particle size, zeta potential, and PDI of

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FX-CN and FX-CS-CN are listed in Table 1. The mean particle diameters of FX-CN and FX-

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CS-CN were both in the nanoscale range, at 237 ± 13 nm and 277 ± 26 nm, respectively. The

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size of FX-CS-CN was significantly larger than that of FX-CN owing to the chitosan coating

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process. The thickness of the chitosan layer could be calculated from the mean particle

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diameter data showing around 40 nm. Similar observations have been reported in many

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papers, showing that applying a chitosan coating onto nanoparticles increases their diameter

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compared with uncoated particles.18,19 The PDI values of FX-CN and FX-CS-CN were 0.31 ±

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0.03 and 0.39 ± 0.02, respectively. The PDI value represents a heterogeneity index. A higher

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PDI indicates that the dispersion state is more heterogeneous, whereas a lower PDI indicates

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that the dispersion state is more homogeneous. Since FX-CN showed a lower PDI value than

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FX-CS-CN did, it could be suggested that FX-CN represents a more homogeneous and stable

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state than FX-CS-CN. Zeta potential is another significant characteristic of nanoparticles and

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indicates the stability of a colloidal suspension. A zeta potential of particles close to an

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absolute value of 30 mV (± 30 mV) has been reported to be a stable state.20,21 In our results,

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the zeta potential of FX-CN was negatively charged (−12.9 ± 1.6 mV) and that of FX-CS-CN

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was positively charged (+24.0± 2.8 mV). Therefore, in terms of colloidal stability, FX-CS-

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CN is more stable than FX-CN. As expected, FX-CN possessed a negative charge because of

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the predominance of ionized carboxylic groups of sodium casein at pH 7.4.21 However,

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chitosan coating induced a change in the surface charge of FX-CS-CN because of the 11 ACS Paragon Plus Environment


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positively charged amine groups of chitosan.22-24

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Different variants of the nanoparticles prepared with varying concentrations of casein in

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the range of 0.5~3% (w/v) were examined to determine the optimal concentration of the

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casein solution to maximize the FX encapsulation efficiency (data not shown). Among them,

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the 2% casein solution demonstrated the best performance for FX encapsulation, and the

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encapsulation efficiencies of FX-CN and FX-CS-CN prepared with the 2% casein solution

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were 73.7% and 71.8%, respectively. Although mechanical processing steps such as

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ultrasonic homogenization and electrospraying can negatively affect the encapsulation

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efficiency, these results showed that the use of casein and chitosan yielded a good

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encapsulation efficiency and a high stability of the nanoparticles.

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After freeze-drying, fine powders of FX-CN and FX-CS-CN with a yellow color were

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obtained, as shown in Figure 1B. The surface morphologies of these two nanoparticles were

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visualized by FE-SEM and presented in Figure 2A and 2B, respectively. The morphologies of

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FX-CN and FX-CS-CN were observed as almost spherical and uniform. The surface of FX-

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CS-CN was smoother than that of FX-CN, which is due to the effect of coating. One of the

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advantages of preparing nanoparticles with casein and chitosan is that the water solubility of

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encapsulated fat-soluble components is improved by these hydrophilic coating materials.24

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Figure 2C presents a comparison of the visual appearances of the two FX-loaded

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nanoparticles and FX-fr added to water. FX-CN and FX-CS-CN showed no sedimentation,

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indicating that FX encapsulated by casein and chitosan was well dispersed in water. However,

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FX-fr was not dispersed and several aggregates were observed on the wall and bottom of the

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tube, indicating the insolubility of FX-fr in water. Figure 2D and 2E showed FX stability in

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the FX-CN and FX-CS-CN during 4 weeks of storage in water with different temperatures. In

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our previous data,25 stability data of free FX in water was significantly decreased according to

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temperature increase. Free FX in water was degraded to almost 30% of initial amount at 26 ℃ 12 ACS Paragon Plus Environment

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after 4 weeks indicating unstable characteristic of free FX under aqueous condition. However,

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more than 80% of initial FX in two nanoparticles was maintained under same aqueous

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condition. Thus, we could confirm the increased stability of FX in the nanoparticles under

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aqueous condition.

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FT-IR analysis was performed to evaluate the interaction between chitosan and the FX-

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loaded casein nanoparticles. Figure 3 shows FT-IR spectra in the wavelength range of

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600−4000 cm-1 for FX-fr, casein, FX-CN, chitosan, and FX-CS-CN. Figure 3A shows the

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spectrum of FX-fr. The peak at 1929.99 cm-1 was assigned as an allenic bond (C=C=C),

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which is considered a functional group of FX.26,27 The peak at 3367.68 cm-1 was assigned as

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hydrogen bonded O–H stretching vibrations. Peaks at 2924.06 cm-1 and 2854.62 cm-1 showed

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the presence of alkanes with C–H bonds. Absorption at 1732.06 cm-1 means the presence of

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ketones with –C=O bonds. Absorption peaks at 1456.24, 1433.80, 1406.09, and 1367.52 cm-1

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were assigned as scissoring and bending alkanes with –C–H bonds. The peak at 1031.91 cm-1

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was characteristic of esters with –C–O bonds. All of these peaks are generally found in FX.27

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Figure 3B shows the spectrum of the casein sodium salt. The peaks at 1637.55 cm-1 and

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1539.18 cm-1 showed the carbonyl group (C=O). Figure 3C shows the spectrum of FX-CN.

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The major absorption peaks shown in Figures 3A and 3B were also found in this spectrum,

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indicating that the FX in FX-fr was well encapsulated within casein in FX-CN. Figure 3D

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shows the spectrum of chitosan. For native chitosan, two peaks at 1649.05 cm-1 and 1556.18

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cm-1 were assigned to the carbonyl bonds (C=O) of the amide group and protonated amine

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group (NH3+).28,29 Figure 3E shows the spectrum of FX-CS-CN, and the combination

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absorption peaks of Figures 3C and 3D could be found in Figure 3E. Taking all of the results

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obtained by FT-IR analysis together, it was concluded that FX from FX-fr has been

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successfully encapsulated in casein and chitosan.

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Stability and bioaccessibility of FX under in vitro digestion assay. Two FX-loaded

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nanoparticles (FX-CN and FX-CS-CN) and Pt-powder were used for an in vitro digestion

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assay to evaluate their digestive stability and bioaccessibility (release from the food matrix)

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during the simulated digestion process of the human GI tract from the mouth to the small

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intestine. In this assay system, each sample was serially treated with the proper enzymes,

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temperature, time, and pH to simulate the five digestion stages, comprising those occurring in

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the mouth, stomach, and three stages of the small intestine (duodenum, jejunum, and ileum)

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in a single test tube. The collected whole digestion solutions from each stage were analyzed

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to estimate the digestive stability of total FX (FX and FXOH), and the micelle phase obtained

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by centrifugation of the solution was analyzed to evaluate the bioaccessibility of total FX

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(Figure 4).

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As shown in Figure 1A, a number of studies have reported that FX is metabolized to

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FXOH by lipase and cholesterol esterase in the small intestine.30 This phenomenon was also

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found with the two types of FX-loaded nanoparticles and Pt-powder during the in vitro

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digestion assay (Figure 4). Since FXOH has an antiobesity activity and FX is primarily

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metabolized to FXOH,30 total FX (FX + FXOH) was measured to determine the stability of

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FX in the in vitro digestion assay. FXOH started to appear after adding pancreatic lipase (and

312

cholesterol esterase) at the beginning stage of the small intestine (duodenum) and the level of

313

transformation from FX to FXOH was slightly increased during the small-intestine stages

314

(duodenum to ileum) according to the increasing treatment time and pH. However, no FXOH

315

was produced before the small-intestine stages of digestion, indicating that this

316

transformation was exclusively mediated by an enzymatic reaction.

317

As a carotenoid, FX can be easily degraded by low pH and oxygen.1 During the digestion

318

process in the GI tract, FX is exposed to these factors by gastric acid and oxidative chemicals.

319

Thus, the stability of FX during the digestion process is an important concern when 14 ACS Paragon Plus Environment

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320

developing new food materials. The analysis of the digestive stability of FX in the two types

321

of nanoparticles and Pt-powder demonstrated that total FX was slightly degraded during the

322

simulated digestion process (Figures 4A–4C). However, if the amount of FX in food is

323

denoted as 100%, the total FX levels were over 80% at the end stage of the small intestine

324

(ileum). This result indicates that most of the FX in the two types of nanoparticles can be

325

stable in either the FX or FXOH form, and had similar stability to that of Pt-powder during

326

digestion in the GI tract.

327

During the digestion process in vivo, mixed micelles and vesicles can solubilize lipids and

328

lipid digestion products from the surface of lipid droplets and are transported to the intestinal

329

epithelium cells coated with the mucous layer for absorption.31 In the in vitro digestion assay,

330

the micelle phase contains these mixed micelles and vesicles, and thus, the FX

331

bioaccessibility of the two types of nanoparticles and Pt-powder can be deduced by analyzing

332

the amount of total FX in this phase. The results presented in Figures 4D–4F showed that the

333

two types of nanoparticles yielded mixed micelles and vesicles containing FX in the micelle

334

phases isolated before the small intestine stages of digestion, while Pt-powder yielded almost

335

no mixed micelles and vesicles containing FX. In the small intestine stages of digestion, the

336

micelle phases of FX-CN and FX-CS-CN contained total FX levels in the ranges of 64–73%

337

and 36–61% of the initial level, respectively. Those levels were higher than that of Pt-powder

338

(26–48%), indicating that the two types of nanoparticles both yield a better bioaccessibility of

339

FX than Pt-powder does and FX can be released more easily from their matrices. In addition,

340

a higher content of FXOH was detected in the micelle phases of the two types of

341

nanoparticles than in Pt-powder. When comparing between FX-CN and FX-CS-CN, both the

342

total FX and FXOH contents in the micelle phase of FX-CN were higher than those in the

343

same phase of FX-CS-CN.

344

A higher bioaccessibility (i.e., a higher release from the food matrix during digestion) 15 ACS Paragon Plus Environment


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345

could be expected to result in a higher bioavailability (a higher absorption and entry into the

346

systemic blood circulation). Besides bioaccessibility, however, two other factors that can

347

influence bioavailability should also be considered. One of these factors is the transport of the

348

released lipophilic components to the intestinal epithelium cells through the mucous layer,

349

and the other factor is the metabolism of the lipophilic components during their absorption

350

and entry into the systemic circulation.31 Consequently, it is too early to conclude that a

351

higher amount of FX or FXOH in the micelle phase of FX-CN indicates more absorption of

352

these molecules into the blood circulation. Nevertheless, it is certain that a higher

353

bioaccessibility is beneficial for achieving a higher bioavailability.

354 355

Bioavailability of FX under in vivo pharmacokinetic and mucin adsorption studies. To

356

evaluate the in vivo absorption rate of FX or FXOH, an in vivo pharmacokinetic study was

357

conducted by feeding a single dose of each sample powder (FX-CN, FX-CS-CN, and Pt-

358

powder) corresponding to 7 mg FX/kg body weight by oral administration to C57BL/6 mice.

359

Among the three metabolites (FX, FXOH, and amarouciaxanthin A) derived from FX in

360

mouse plasma samples, only FXOH was analyzed by LC-MS/MS because it is the primary

361

metabolite

362

amarouciaxanthin A, the metabolite of FXOH in mouse liver, has not been reported in human

363

plasma, and thus, it remains uncertain whether this compound is a metabolite of FX in human.

364

After administration, plasma samples were collected at 1, 2, 4, 8, and 24 h, and

365

pharmacokinetic parameters such as Tmax, Cmax, T1/2, AUCt, and AUC∞ were calculated.

of

FX

detected

most

abundantly

in

mouse

plasma30.

Furthermore,

366

Figure 5A shows the FXOH concentration in plasma and Table 2 shows the

367

pharmacokinetic data of FXOH for the two types of nanoparticles (FX-CN and FX-CS-CN)

368

and Pt-powder. The time (Tmax) at which the maximum plasma concentration (Cmax) was 16 ACS Paragon Plus Environment

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369

detected was 2.7 h for Pt-powder and over 4.7 h for the two types of nanoparticles. This result

370

indicates that the two types of nanoparticles reached the blood circulation later than Pt-

371

powder through the intestinal epithelium cells even though bioaccessibility (release from the

372

food matrix) of each type of nanoparticle was higher than that of Pt-powder. The

373

bioavailability of FXOH can be deduced from the area under the plasma concentration-time

374

curve to infinity (AUC∞). As shown in Table 2, the AUC∞ of FX-CS-CN was 531 nmol·h/L,

375

which is almost two times higher than that of FX-CN (276 nmol·h/L) and Pt-powder (280

376

nmol·h/L). Therefore, it can be concluded that FX-CS-CN has a higher bioavailability than

377

the other two samples do and FX-CN has a similar bioavailability to that of Pt-powder.

378

The pharmacokinetic bioavailability data shown in Figure 5A and Table 2 showed quite

379

different results from those of the bioaccessibility data from the previous in vitro digestion

380

assay. There might be several factors influencing the differences between the results of these

381

experiments, and one of them could be the chemical characteristics of two nanoparticles.

382

Among the tested characteristics in Table 1 and Figures 2 and 3, the most significant

383

difference between these two nanoparticles was their opposite zeta potentials due to the

384

chitosan coating process. This factor may affect the absorption ratios of these two types of

385

nanoparticles through the intestinal epithelium cells. Since the epithelium cells in the small

386

intestine are coated with a mucous layer that is the first barrier for nutrients, and this layer

387

possesses a negative charge,31 FX-CS-CN with a positive charge is likely to be adsorbed for

388

longer on this mucous layer by electrostatic force than FX-CN with a negative charge. In

389

order to investigate this property, a mucin adsorption study was carried out with the two types

390

of nanoparticles, and the results showed that FS-CS-CN had a higher mucin adsorption level

391

(61.2%) than FX-CN (34.7%), as expected (Figure 5B). Interactions between FX-CS-CN and

392

mucin may be caused by electrostatic interactions between the amine group (NH3+) of 17 ACS Paragon Plus Environment


393

chitosan and the carboxyl group (COO−) of mucin. Consequently, FX-CS-CN demonstrated a

394

higher bioavailability than FX-CN did, even though the bioaccessibility of FX-CS-CN was

395

lower than that of FX-CN, and the positive zeta potential induced by chitosan coating was

396

suggested as one of the main factors underlying that difference.

397

In conclusion, two types of FX-loaded nanoparticles (FX-CN and FX-CS-CN) were

398

developed using casein and chitosan as encapsulation materials with an FX-enriched fraction

399

extracted from the microalga P. tricornutum. These two new materials showed typical

400

characteristics of such nanoparticles, including size, zeta potential, PDI, morphology, and the

401

improved water solubility. In addition, FX-CS-CN yielded a better bioavailability of FXOH

402

than FX-CN and Pt-powder. Chitosan may also affect the tight junction integrity and cell

403

permeability. These results indicate that FS-CS-CN can be a good material for the application

404

of FX with improved water solubility and bioavailability in the food industry.

405 406

Financial support

407

This research was supported by a grant from Marine Biotechnology Program (2MP0360)

408

funded by Ministry of Oceans and Fisheries, Korea and an intramural grant (2Z04690) from

409

KIST Gangneung Institute of Natural Products

410 411

Note

412

The authors declare no competing financial interest.

413 414

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523 524 525



Figure captions

Figure 1. Fucoxanthin (Fx) metabolism during digestion process (A) and the preparation process of FX-loaded nanoparticle from P. tricornutum powder (Pt-powder) based on casein and chitosan (B).

Figure 2. Field emission scanning electron microscope (FE-SEM) images of FX-CN (A) and FX-CS-CN (B) with a scale bar of 500 nm. At the concentration of 3 mg/mL, these nanoparticles dissolved in water showed good dispersion, whereas FX-fr was not fully dissolved in water (C), and fucoxanthin in FX-CN (D) and FX-CS-CN (E) was maintained over 80% of initial amount in the aqueous solution during 4 weeks storage period.

Figure 3. Fourier transform-infrared (FT-IR) spectrums of a fucoxanthin (FX)-enriched fraction (FX-fr) from P. tricornutum (A), casein (B), FX-casein nanoparticles (FX-CN) (C), chitosan (D), and chitosan-coated FX-CN (FX-CS-CN) (E). Each FT-IR spectrum was analyzed in the wavelength range of 4000–600 cm-1.

Figure 4. Digestive stability (A–C) and bioaccessibility (D–F) of fucoxanthin (FX) in FX-CN, FX-CS-CN, and Pt-powder during an in vitro simulated digestion assay. From the duodenum stage of digestion, FX started to be transformed to FXOH by lipase and cholesterol esterase. The difference numbers and letters represent significant differences of means.

Figure 5. (A) Pharmacokinetic data of two types of fucoxanthin (FX)-loaded nanoparticles and P. tricornutum powder (Pt-powder). A single dose of each sample powder was orally 24 ACS Paragon Plus Environment

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administrated to C57BL/6 mice, and fucoxanthinol (FXOH), which is a primary metabolite of FX, was analyzed in plasma samples collected at several time points during 24 h after the administration. Pt-powder was used as a control. (B) Mucin adsorption characteristics of the two types of FX-loaded nanoparticles. Asterisk indicates significant difference at P < 0.001.



Page 26 of 33

Tables

Table 1. Characteristics of FX-CN and FX-CS-CN. Different Asterisks Indicate Significant Differences (*, P < 0.5; **, P < 0.1).

Nanoparticle

Size

Zeta potential

PDI

Encapsulation

(nm)

(mV)

FX-CN

237 ± 13

–12.9 ± 1.6**

0.31 ± 0.03*

73.7 ± 9.0

FX-CS-CN

277 ± 26

24.0 ± 2.8

0.39 ± 0.03

71.8 ± 2.2

efficiency (%)


Page 27 of 33


Table 2. Pharmacokinetic Parameters for Fucoxanthinol (FXOH) from the Plasma Samples of C57BL/6 Mice Orally Administered with FX-CN, FX-CS-CN, and Pt-powder in a Single Dose Mode. The Difference Letters Represent Significant Differences of Means.

Pharmacokinetic parameters Sample

Tmax

Cmax

T1/2

AUCt

AUC∞

(h)1

(nmol/L)2

(h)3

(nmol·h/L)4

(nmol·h/L)5

a

FX-CN

5.3 ± 2.3

FX-CS-CN

4.7 ± 3.1

Pt-powder

2.7 ± 1.2

ab

b

23.7 ± 9.1

b

48.6 ± 16.2 16.2 ± 8.1

a

ab

b

4.4 ± 0.9 5.1 ± 1.1

ab

6.8 ± 3.3

a

b

324 ± 37

509 ± 134 219 ± 125

1

Cmax: Maximum plasma concentration

2

Tmax: Time for Cmax

3

T1/2: Terminal half-life

4

AUCt,: Area under the plasma concentration-time curve

5

AUC∞: Area under the plasma concentration-time curve to infinity


276 ± 45 a

b

b

532 ± 121 280 ± 53

a

b


Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5


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The Table of Contents Graphic


Preparation of Fucoxanthin-Loaded Nanoparticles Composed of

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