In Vitro Bioavailability, Cellular Antioxidant Activity, and Cytotoxicity of

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In vitro bioavailability, cellular antioxidant activity, and cytotoxicity of #-carotene-loaded emulsions stabilized by catechin-egg white protein conjugates Luping Gu, Che Pan, Yujie Su, Ruojie Zhang, Hang Xiao, David Julian McClements, and Yan-Jun Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05909 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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Journal of Agricultural and Food Chemistry

In vitro bioavailability, cellular antioxidant activity, and cytotoxicity of β-carotene-loaded emulsions stabilized by catechin-egg white protein conjugates Luping Gu†,‡, Che Pan‡, Yujie Su†, Ruojie Zhang‡, Hang Xiao‡, David Julian McClements*,‡, Yanjun Yang*,†

†

Key Laboratory of Food Science and Technology, School of Food Science and Technology,

Jiangnan University, Wuxi 214122, People’s Republic of China ‡

Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States

*Corresponding author: Email address: [email protected]; [email protected]

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Abstract

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Previously, it was shown that catechin-egg white protein (CT-EWP) conjugates were

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effective antioxidant emulsifiers that could form and stabilize emulsions, and also inhibit the

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degradation of encapsulated carotenoids. The objective of the current study was to evaluate the

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impact of conjugation on the in vitro bioavailability, cellular antioxidant activity, and cytotoxicity

6

of β-carotene-loaded emulsions. Lipid droplets coated with EWP or with CT-EWP conjugates

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exhibited quite similar behavior when they were passed through a simulated gastrointestinal

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tract. The β-carotene encapsulated in emulsions stabilized by CT-EWP conjugates exhibited a

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higher overall in vitro bioavailability, which was attributed to a greater stability of the

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carotenoids to chemical transformation. The emulsions stabilized by CT-EWP conjugates also

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exhibited greater ability in inhibiting oxidation in a cell-based assay (dichlorofluorescin

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diacetate). Cytotoxicity analysis suggested that β-carotene emulsions stabilized by CT-EWP

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conjugates only exhibited a slight cytotoxicity when used at high concentrations. These results

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suggest that CT-EWP conjugates can be used to formulate emulsion-based delivery systems for

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chemically labile hydrophobic bioactives with enhanced antioxidant activity and bioavailability.

16 17

Keywords: β-carotene; conjugates; bioavailability; antioxidant activity; cytotoxicity


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Introduction Some carotenoids, such as β-carotene, have been shown to be bioactive components that 1, 2

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have potential health benefits

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vitamin A activity and acts as an effective antioxidant

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highly hydrophobic and chemically labile molecule in functional foods is currently restricted due

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to its poor water-solubility, chemical instability, and low oral bioavailability 5. These limitations

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can be overcome by encapsulating β-carotene within well-designed emulsion-based delivery

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systems, such as emulsions or nanoemulsions 6, 7. In O/W emulsions, β-carotene is present inside

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small oil droplets that are dispersed in a water phase. The oil droplets are coated by a layer of

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emulsifier molecules, which are usually selected to ensure good physical stability of the

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emulsions by inhibiting flocculation or coalescence, but which may also be selected to provide

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protection against chemical instability (such as oxidation). After ingestion, it is important that the

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encapsulated carotenoids have a high oral bioavailability, which depends on their

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bioaccessibility, transformation, and absorption characteristics 7. First, the lipid droplets should

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be fully digested and release the encapsulated carotenoids into the surrounding intestinal fluids in

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an absorbable form (bioaccessibility). In the stomach and small intestine, the triacylglycerol

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molecules at the surfaces of the oil droplets are hydrolyzed by lipase into free fatty acids and

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monoacylglycerols, which then mix with bile salts and phospholipids to form mixed micelles

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capable of solubilizing and transporting the β-carotene molecules released from the lipid

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droplets. In addition, the efficacy of the ingested carotenoids depends on the fraction that

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remains in a biologically active form (transformation) and the fraction that is transported across

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the epithelium cells (absorption). Consequently, it is important to determine the impact of any

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new emulsifier used to fabricate an emulsion-based delivery system on the oral bioavailability of

. Numerous studies have reported that β-carotene has high pro3, 4

. Nevertheless, the utilization of this



41 42

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encapsulated nutraceuticals. Recently, it has been shown that antioxidant emulsifiers can be fabricated by covalently

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attaching polyphenols to proteins 8. The proteins provide good emulsifying properties

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whereas the polyphenols provide strong interfacial antioxidant activity

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have shown the protein-polyphenol conjugates synthesized using a free radical grafting method

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improved the physical and chemical stability of β-carotene-enriched emulsions, when compared

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with native proteins or conjugates prepared using other approaches

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catechin-egg white proteins (CT-EWP) conjugates were fabricated using free radical grafting and

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then the conjugates were utilized as a novel antioxidant emulsifier to prepare β-carotene-enriched

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emulsions. Compared to native proteins, CT-EWP conjugates formed emulsions with improved

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stability to environmental stresses (such as pH, salt, and heating), and that were better at

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inhibiting the degradation of encapsulated β-carotene during storage. However, to the best our

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knowledge, no information is currently available about the impact of these conjugates on the

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bioavailability of the encapsulated bioactive agents.

14, 15

11-13

9, 10

,

. Previous studies

. In our previous study,

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Consequently, a major objective of the current study was to determine the impact of

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stabilizing emulsions with CT-EWP conjugates on the potential oral bioavailability of

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encapsulated β-carotene. As mentioned earlier, the overall bioavailability depends on the

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bioaccessibility, transformation, and absorption of the bioactive within the gastrointestinal tract

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(GIT). The bioaccessibility and transformation of the carotenoids was determined using a three-

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stage simulated gastrointestinal fate (GIT) model consisting of mouth, stomach, and small

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intestine phases 16. The absorption of the carotenoids was determined using a cell culture model

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(Caco-2 cells) that mimics the human intestinal layer

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EWP conjugates had strong antioxidant activity, but the in vitro chemical assays used in that

17, 18

. Our previous work found that CT-


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study have low relevance for predicting the potential antioxidant effects of nutraceuticals within

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the human body. For this reason, the cellular antioxidant activity of the conjugates and β-

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carotene emulsions was also analyzed in the current study. Moreover, the potential toxicity of the

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CT-EWP conjugate-stabilized emulsions was tested to evaluate their potential for applications in

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the food industry.

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The objective of this study was to evaluate the bioavailability, cellular antioxidant activity,

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and cytotoxicity of β-carotene emulsions stabilized by CT-EWP conjugates so as to establish the

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potential of this new antioxidant emulsifier for application in functional foods and beverages.

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Materials and Methods

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Materials

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Fresh eggs and sunflower oil were purchased from a local supermarket. β-carotene, 2’,7’-

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dichlorofluorescin diacetate (DCFH-DA), tert-Butyl hydroperoxide (t-BHP, 70%), 3-(4,5-

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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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isothiocyanate (FITC), mucin from porcine stomach, pepsin from porcine gastric mucosa, lipase

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from porcine pancreas, and porcine bile extract were purchased from Sigma-Aldrich (St. Louis,

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MO, USA). Human colon carcinoma cell lines (Caco-2 cells, ATCC, Manassas, VA) were used

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for cellular cytotoxicity, antioxidant activity and uptake assays after 30~45 passages. Dulbecco’s

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modified Eagle’s medium (DMEM) (with 4.5g/L Glucose, L-Glutamine, and Sodium Pyruvate),

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fetal bovine serum (FBS), penicillin-streptomycin (10,000 U/mL), Non-Essential Amino Acids

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Solution (NAA, 100 X), HEPES (1 M), Trypsin-EDTA (0.25%), Phosphate Buffered Saline (10

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X), Methyl tert-butyl ether (MTBE), n-hexane, chloroform, Dimethyl sulfoxide (DMSO) were

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obtained from Fisher Scientific (Agawam, MA, USA). All other chemicals and reagents used

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were analytical grade. Double distilled water was used to prepare all aqueous solutions.

bromide

(MTT),

Nile

red,

fluorescein



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Preparation of β-carotene Emulsions

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Catechin-egg white protein (CT-EWP) conjugates were synthesized using a free radical

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grafting method, and the amount of catechin bound to the egg white protein was found to be 5.71

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mg/g conjugates. Their successful fabrication was confirmed using electrophoresis and liquid

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chromatography-mass spectrometry, as described in our previous study

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conjugates were used as an antioxidant emulsifier to encapsulate β-carotene within oil-in-water

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emulsions. In brief, an aqueous phase was prepared by dissolving CT-EWP conjugates (2%,

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w/w) in phosphate buffer solution (5 mM, pH 3.0) and then stirring overnight at 25 °C to ensure

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dissolution. An oil phase was prepared by dispersing β-carotene (0.1%) in sunflower oil and then

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sonicating for 20 min at 50 °C in the absence of light to ensure complete dispersion and

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dissolution of the carotenoid crystals. Coarse emulsions were then fabricated by mixing the

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aqueous phase (90% w/w) and oil phase (10% w/w), using a high-shear mixer for 2 min

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(M133/1281-0, Biospec Products, Inc., ESGC, Switzerland). After that, the coarse emulsions

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were passed through a high-pressure homogenizer (M110P, Microfluidics, Newton, MA) at

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12000 psi for three cycles to form β-carotene-loaded oil-in-water emulsions. After preparation,

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the emulsions were immediately placed in a refrigerator before further use. Emulsions prepared

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using egg white protein (EWP) (rather than CT-EWP conjugates) as an emulsifier were used as

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controls.

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Cellular Antioxidant Activity of β-carotene Emulsions

106

15

. The CT-EWP

The cellular antioxidant of the samples was evaluated in Caco-2 cells using the DCFH-DA 19

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assay described previously

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NAA, and 1% penicillin and streptomycin at 37 °C in an incubator with 5% CO2 and 95%

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humidified air. When the cells reached 80% confluence, they were seeded and grown in 96-well

. Caco-2 cells were cultured in DMEM containing 10% FBS, 1%


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plates (1×105 cells/mL). After 24 hours, the cells were treated with different concentrations of β-

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carotene emulsions stabilized by either CT-EWP conjugates or EWP, or they were treated with

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aqueous solutions containing either CT-EWP conjugates or EWP. The samples were obtained by

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diluting initial emulsions and solutions 20, 40, 60, 80, or 100 times with DMEM and then

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treating the cells for 24 hours. After that, the plates were treated with 100 µL of DCFH-DA (10

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µM) per well and incubated at 37 °C for 30 min. The plates were then washed with PBS (1 X)

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twice before adding 100 µL of t-BHP (500 µM) to each well (except the negative control) and

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then incubating for 30 min at room temperature. Finally, the fluorescence intensity of the

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samples was recorded using a Microplate Reader (Synergy 2 Multi-Mode, BioTek Instruments,

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Inc., Winooski, VT) at an emission wavelength of 528 nm and an excitation wavelength of 485

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nm.

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The cellular antioxidant activity of the samples was then calculated using the following equation: Cellular Antioxdant Activity% =

− !"#$" −

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Here, Asample is the fluorescence intensity of the cells treated with different samples, Ablank is

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the fluorescence intensity of the blank wells, and Acontrol is the fluorescence intensity of the cells

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treated with DMEM only.

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In vitro Cytotoxicity of β-carotene Emulsions

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The MTT assay was utilized to determine the impact of the samples on cell viability and

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therefore evaluate their potential cytotoxicity 20. In brief, Caco-2 cells were incubated in 96-well

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plates at a density of 1×105 cells/mL at 37 °C in 5% CO2 incubator. After incubation for 24

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hours, the cells were treated with β-carotene emulsions stabilized by either CT-EWP conjugates

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and EWP, or with aqueous solutions containing either CT-EWP conjugates or EWP. A range of 7 ACS Paragon Plus Environment


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concentrations was utilized for each sample tested by diluting with DMED (20, 40, 60, 80, and

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100 times) prior to analysis so as to ascertain the influence of emulsifier concentration on

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cytotoxicity. After a further 24 hours, the cells were treated with 100 µL MTT solution (0.5

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mg/mL) and incubated for 1 hour at 37 °C. Then, 100 µL of DMSO was used to dissolve the

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products and the absorbance was measured at a wavelength of 570 nm using a Microplate Reader

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(Synergy 2 Multi-Mode, BioTek Instruments, Inc., Winooski, VT).

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The cell viability was then calculated using the following equation: Cell Viability% =

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− !"#$" −

Here, Asample is the absorbance of the cells treated with samples, Ablank is the absorbance of

140

blank wells, and Acontrol is the absorbance of cells treated with DMEM only.

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In Vitro Digestion of β-carotene Emulsions

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A three-step simulated gastrointestinal tract (GIT) model that consisted of mouth, stomach

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and small intestine phases was utilized to evaluate the potential gastrointestinal fate of the

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different samples and controls. The simulated saliva fluid (SSF) (sodium chloride 1.594 g/L,

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ammonium nitrate 0.328 g/L, potassium phosphate 0.636 g/L, potassium chloride 0.202 g/L,

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potassium citrate 0.308 g/L, uric acid sodium salt 0.021 g/L, urea 0.198 g/L, lactic acid sodium

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salt 0.146 g/L), simulated gastric fluid (SGF) (sodium chloride 2 g/L, hydrochloric acid 83.3

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mM), and simulated intestine fluid (SIF) (calcium chloride dihydrate 36.67 g/L, sodium chloride

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219.13 g/L) were prepared as described as our previous study 21.

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Mouth phase: Initially, 4 g of β-carotene emulsion was mixed with 16 g of phosphate buffer

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(5 mM, pH 3.0) to reach the desired oil concentration (2%, w/w). Then, 20 g of the diluted

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emulsions and 20 g of SSF containing mucin (30 mg/mL) were preheated to 37 °C and then

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mixed together. The mixture was adjusted to pH 6.8 and then swirled for 2 min at a speed of 100 8 ACS Paragon Plus Environment

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rpm in an incubator shaker set at 37 °C (Innova Incubator Shaker, Model 4080, New Brunswick

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Scientific, Edison, NJ).

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Stomach phase: 20 g of the sample resulting from the mouth phase was mixed with 20 g of

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SGF (37 °C) containing pepsin (3.2 mg/mL) and then adjusted to pH 2.5. After that, the mixture

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was placed in the incubator shaker and swirled for 2 h at 100 rpm and 37 °C.

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Small intestine phase: 30 g of the sample resulting from the stomach phase was placed in a

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water bath (37 °C) and adjusted to pH 7.0. Subsequently, 1.5 mL of SIF and 3.5 mL of bile

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extract (53.57 mg/mL) were added, and the mixture was then adjusted back to pH 7.0. Then, 2.5

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mL of lipase suspension (24 mg/mL) was added and the mixture was maintained at pH 7.0 for 2

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h by adding NaOH solution (0.25 M) using a pH-stat automatic titration device (Metrohm USA

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Inc., Riverview, FL, USA). The volume of NaOH solution required to neutralize the FFA was

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recorded during the incubation. The amount of free fatty acids released was then estimated using

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the following equation: FFA% =

()*+ × -)*+ × ./001 × 100 2/001 × 2

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Here, VNaOH is the volume of NaOH solution required to neutralize the FFA, mNaOH is the

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molarity of the NaOH solution (0.25 M), MLipid is the average molecular weight of the sunflower

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oil (876 g/mol), and WLipid is the weight of the sunflower oil in the digestion system (0.15 g).

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Determination of Particle Characterization

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The particle size distribution and mean particle diameter of the samples exposed to different

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regions of the simulated GIT were determined using a laser light scattering instrument

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(Mastersizer 2000, Malvern Instruments Ltd., Malvern, Worcestershire, UK). Prior to particle

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size measurements, the initial emulsions were diluted with phosphate buffer (5 mM, pH 3) to

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minimize multiple scattering effects. The mouth and small intestine samples were diluted with 9 ACS Paragon Plus Environment


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neutral phosphate buffer (5 mM, pH 7), while the stomach samples were diluted with acidic

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phosphate buffer (5 mM, pH 2.5). The ζ-potential values of the particles in the samples were

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determined using a particle electrophoresis device (Zetasizer Nano, Malvern Instruments,

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Worcestershire, UK). In this case, the initial emulsions were diluted with acidic phosphate buffer

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(5 mM, pH 3), the mouth samples were diluted with SSF (pH 6.8), the stomach samples were

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diluted with SGF (pH 2.5), and the small intestine samples were diluted with neutral phosphate

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buffer (5 mM, pH 7). The refractive index of the aqueous phase and oil phase used in the

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calculations were 1.33 and 1.45, respectively.

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For microstructure analysis, the samples exposed to different regions of the GIT were

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collected and then diluted with phosphate buffer as described for the particle size measurements.

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Then, 1 mL of diluted samples was transferred to a glass test tube, and then, 20 µL of Nile red (1

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mg/mL) and 20 µL of FITC (1 mg/mL) were added to stain the lipids and proteins, respectively.

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After that, a drop of sample was placed on a microscope slide and covered with a thin glass

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cover slip. The microstructural images were captured using a Nikon confocal microscope (Nikon

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D-Eclipse C1 80i, Nikon, Melville, NY, USA) at a magnification of ×600 (×60 objective lens and

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×10 eyepiece lens) and then analyzed using image analysis software (NIS-Elements, Nikon,

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Melville, NY).

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Determination of β-carotene Oral Bioavailability

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The overall oral bioavailability (BA) of bioactives is determined by three main factors:

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bioaccessibility (B*); absorption (A*); and stability to transformation (T*), as described

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previously by our research group 7, 22: BA% = 7 ∗ × ∗ × 9 ∗

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For this reason, we carried out experiments to determine the relative contribution of these


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three factors to the overall bioavailability of β-carotene in the emulsion-based delivery systems

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prepared in this study.

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Bioaccessibility (B*): After emulsions had passed through the three-step simulated GIT

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model, 20 g of small intestine sample was centrifuged (18,000 rpm, Thermo Scientific, Waltham,

202

MA) at 4 °C for 30 min. The yellow supernatant that appeared after centrifugation of the samples

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was assumed to be the “micelle” fraction in which the β-carotene was solubilized, and the

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carotenoid concentration in this fraction was determined according to a method described

205

previously 23. In brief, 2 mL of micelle phase was mixed with 2 mL of chloroform and 2 mL of

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ethanol, and then the mixture was shaken vigorously for 20 s and centrifuged for 5 min at 3500

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rpm. The supernatant layer was then transferred to a glass test tube and diluted with chloroform

208

to an appropriate concentration. Then, the absorbance of the diluted supernatant was recorded at

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450 nm using a UV-visible spectrophotometer (Cary 100 UV-Vis, Agilent Technologies, Santa

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Clara, CA, USA), with chloroform as a control. The β-carotene concentration in the total small

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intestine sample (without centrifugation) was also measured using the same approach. The

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carotenoid concentrations were determined from the absorbance measurements using a standard

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curve prepared using samples of known β-carotene level. The bioaccessibility of β-carotene was

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calculated using the following equation: Bioaccessibility% =

-;0! × 100 -

In Vitro Bioavailability, Cellular Antioxidant Activity, and Cytotoxicity of

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