Effects of Lipids on in Vitro Release and Cellular Uptake of Î²-Carotene

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Effects of lipids on in vitro release and cellular uptake of #-carotene in nanoemulsion-based delivery system Jiang Yi, Fang Zhong, Yuzhu Zhang, Wallace Yokoyama, and Liqing Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04789 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 4, 2015

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Journal of Agricultural and Food Chemistry

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Effects of lipids on in vitro release and cellular uptake of β-carotene in nanoemulsion-

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based delivery system

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Jiang Yi1*, Fang Zhong2, Yuzhu Zhang3, Wallace Yokoyama3, Liqing Zhao1*

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and Technology, Jiangnan University, Wuxi 214122, China

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College of chemistry and environmental engineering, Shenzhen University, Shenzhen 518060, China Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Food Science

Western Regional Research Center, ARS, USDA, Albany, California 94710, United States

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*To whom correspondence should be addressed.

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Tel (J.Y.): 86-755-26557377. Fax: 86-755-26536141. E-mail: [email protected];

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Tel (L.Z.): 86-755-26733095. Fax: 86-755-26536141. E-mail: [email protected].

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ACS Paragon Plus Environment


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Abstract:

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β-carotene (BC) nanoemulsions were successfully prepared by microfluidization. BC

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micellarization was significantly affected by bile salts and pancreatin concentration.

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Positive and linear correlation was observed between BC release and bile salts

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concentration. Pancreatin facilitated BC’s release in simulated digestion. Compared to

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the control (bulk oil) (4.6%), nanoemulsion delivery systems significantly improved the

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micellarization of BC (70.9%). The amount of BC partitioned into micelles was positively

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proportional to the length of carrier oils. Unsaturated fatty acid (USFA)-rich oils were

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better than saturated fatty acid (SFA)-rich oils in transferring BC (p0.05). A positive and linear relationship

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between the degree of lipolysis and the release of BC in vitro digestion was observed.

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Bile salts showed cytotoxicity to Caco-2 cells below 20 times dilution. BC uptake by

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Caco-2 cells was not affected by fatty acid (FA) compositions in micelles, but BC uptake

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was proportional to its concentration in diluted micelles fraction. The results obtained

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are beneficial to encapsulate and deliver BC or other bioactive lipophilic carotenoids in a

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wide range of commercial products.

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Keywords: β-carotene, lipids, bioaccessibility, Caco-2 cell

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Introduction

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β-carotene (BC) is one of the most highlighted naturally occurring carotenoids for

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human health due to its high pre-vitamin A activity and strong antioxidant activity.1, 2

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Epidemiologic research showed that BC has the ability to inhibit cancer, prevent age

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related macular degeneration and reduce the risk of cardiovascular diseases.3, 4 BC is

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also the precursor of ligands necessary for the activity of nuclear receptors that regulate

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energy metabolism. Recently, some studies have shown that BC reduces weight gain

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and may have an effect on obesity.5 Besides, as an excellent colorant, BC is widely used

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in beverage industry.6 The color varied from yellow to orange red depending on BC

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content.

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However, the utilization of BC is strictly restricted due to its low solubility in water

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and high susceptibility to isomerization, oxidation, and degradation by light, oxygen, and

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heat. Compared to conventional emulsions, nanoemulsions are better choices to deliver

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BC with many potential advantages (such as, better bioavailability and higher stability).7

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Numerous studies on the physicochemical stability of BC in nanoemulsions have been

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reported, but information that impacted BC’s bioaccessibility is lacking.8-10 Many factors

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including particle size, carriers and emulsifiers influence the bioaccessibility of

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encapsulated BC.11, 12 The bioavailability improved with the decrease of particle size,11, 13

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and BC absorption was affected by dietary oils.14 Plenty of studies illustrated that dietary

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fat could promote BC absorption,

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carotenoids in vegetable salad with full-fat dressing were higher than that with a

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reduced-fat salad dressing.19

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compared to no oils. The bioavailability of

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In addition, BC bioavailability was also impacted by the characteristics of dietary oils.

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In vitro, the bioavailability of BC in salad meal incorporated into micelles were raised by

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adding long chain triglycerides (LCT) compared to medium chain triglycerides (MCT).16 In

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humans, there is an increase in BC bioavailability when LCT were added compared to

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MCT.20 BC bioaccessibility was relatively higher in corn oil (66%) than in MCT

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nanoemulsion (2%).21 The small mixed micelles formed through MCT digestion may be

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the main reason for the low BC bioaccessibility. The authors also demonstrated the

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modulation of BC bioaccessibility by controlling oil composition and concentration in

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nanoemulsion delivery systems.22 The saturation degrees of oils used in nanoemulsions

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may also impact the transfer of BC. However, to date, no such study was reported.

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A recent study illustrated that compared with oils rich in monounsaturated fatty acid

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(MUFA) and polyunsaturated fatty acid (PUFA), dietary oils rich in saturated fatty acid

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(SFA) leaded to a higher bioavailability of lutein and zeaxanthin.23 Nevertheless, a study

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in humans showed that lutein and α-carotene absorption was better in MUFA rich

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canola oil than in SFA rich butter.24 These contradictory findings highlighted the need for

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additional investigations.

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In this research, six dietary lipids (corn oil, canola oil, olive oil, palm oil, coconut oil,

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and MCT) based on their distinctly different fatty acid compositions (Table 1) were

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selected as BC carriers and six BC nanoemulsions were prepared. The composition of six

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lipids was analyzed by saponification with ethanolic KOH solution (1.0M KOH in 90%

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ethanol) followed by fatty acyl methylation in the presence of 20% 1,1,3,3-

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tetramethylguanidine

(TMG)/methanol

(v/v)

4

solutions


and

detected

by

gas

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chromatography.25 Reboul et al.26 showed that the results obtained about carotenoids

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uptake with in vitro models were well correlated with in vivo absorption studies. In this

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study, both in vitro simulated gastrointestinal digestion and cellular uptake by Caco-2

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cells model were used for screening BC bioavailability. The goal of this study was to

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obtain a better understanding of the effect of oils on BC bioaccessibility in

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nanoemulsions.

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Materials and Methods

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Materials

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Corn oil, canola oil, and olive oil were purchased from a local market (Albany, CA,

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U.S.). Neobee 1053 (medium-chain triglycerides, MCT) was kindly supplied by Stepan

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Company (Maywood, New Jersey, U.S.). Organic coconut oil and palm oil were donated

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by Ciranda Company (Hudson, Wisconsin U.S.). Sodium caseinate (Alanate 180) was

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purchased from Fonterra Co-operative Group (Auckland, New Zealand). β-carotene (97%,

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#22040), pepsin (porcine, P6887), bile extract (porcine, B8631), pancreatin (porcine

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pancreas, P7545), and HPLC-grade solvents (methanol, ethanol, acetonitrile,

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dichloromethane, n-hexane) were purchased from Sigma-Aldrich (St. Louis, MO U.S.)

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and used without further purification. Dulbecco’s modified Eagle’s medium (DMEM)

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(containing 4.5 g/L D-glucose and GlutaMAX™), penicillin and streptomycin (100×), fetal

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bovine serum (FBS), TrypLETM Select, Hanks’ balanced salt solution (HBSS), and

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phosphate buffer solution (PBS)(10×) were purchased from GIBCO (Grand Island, NY,

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U.S.). The human colon carcinoma cell line (Caco-2 cells, ATCC, Manassas, VA, U.S.) were

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grown in DMEM. Cells after 60-80 passages were used in the uptake study. Analytical

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grade hydrochloric acid and sodium hydroxide, sodium chloride and calcium chloride

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were purchased from Fisher Scientific (Fair Lawn, NJ, U.S.). Ultrapure water was used in

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all experiments.

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Methods

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Preparation of β-carotene emulsions

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Sodium caseinate (SC) was dispersed in ultrapure water and stirred for 3 h to form a 2%

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solution. β-carotene (BC, 0.1%) was dissolved in carrier oils by stirring for 5 min at 50 °C,

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and continued to stir for 1 h at room temperature to ensure complete solubilization

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under nitrogen. Crude emulsions of 10 wt% of the BC vegetable oils solution in the 2%

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SC solution at a mass ratio of 1:9 (oil phase:aqueous phase) was formed by high-speed

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homogenization for 2 min (PT10/35, Kinematica GmbH, Switzerland). The crude

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emulsions were then further homogenized through a high pressure microfluidizer (M-

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110L, Microfluidics, MA, U.S.) seven times at 15000 psi (103.4 MPa) at 37 °C. After the

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preparation, all stock samples were put in a refrigerator (2–6 °C) for subsequent uses.

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Particle diameter analysis

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The mean particle sizes (Dz), polydispersity indices (PDI), and Z-potential were

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determined by dynamic light scattering (Zetasizer Nano, Malvern Instruments,

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Worcestershire, UK).

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samples 1:100 with pH-adjusted ultrapure water (pH 7.0). The refractive index values

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used for the instrumental analysis of oil droplets and dispersant were 1.45 and 1.33,

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respectively. All measurements were made in triplicate and were conducted at 25 °C.

Nanoemulsion samples were prepared by diluting the stock

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In vitro digestion

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A two-stage in vitro digestion method was used to simulate the conditions of human

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stomach and intestine as described by Garrett et al..27 At simulated gastric digestion

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phase, 7.5 mL sample was mixed with 10 mL gastric fluid (3.2 mg/mL pepsin and 0.15 M

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NaCl) in a water jacketed beaker at 37 °C (Thermo Scientific, NH, U.S.). The pH was

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adjusted to 2.0 with 2.5 M HCl and 0.1 M NaOH and incubated with stirring at 250 rpm

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for 1 h. For simulated intestinal digestion, the samples were immediately adjusted to pH

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7 with 0.5 M NaOH solution. Fifteen mL of simulated intestinal fluid containing 10 mM

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CaCl2 with different concentrations of bile extract (0.0-20 mg/mL) and pancreatin (0.0 -

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2.4 mg/mL) at pH 7 was added to the digestion samples to facilitate lipolysis and BC

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micellarization. Final concentrations of bile extract and pancreatin were optimized to be

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20mg/mL and 1mg/mL, respectively. The acidity increased immediately due to the lipase

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hydrolysis of the emulsified triglycerides and the pH was maintained at 7 by addition of

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0.25 M NaOH using an autotitrator (TitraLab TiM840, Radiometer, Lyon, France). The

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amount of NaOH added over time (120 min) was recorded. The temperature was kept at

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37 °C with a thermostatic water bath and stirred at 250 rpm. The extent of lipolysis was

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calculated from the volume of NaOH solution added at each time point by the following

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equation.28

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Where VOLNaOH is the volume of NaOH added. And ConcNaOH is 0.25 in this case.

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Moletriglyceride was the average molecular weight of the triglycerides of vegetable oils. The

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average molecular weight was calculated from: 7



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Where Saponification value (SV) of MCT, coconut oil, palm oil, olive oil, canola oil, and

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corn oil are 334, 252, 195, 188, 187, and 190, respectively. MwKOH is the molecular

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weight of KOH, 56 g/mol.

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After in vitro digestion an aliquot of the digesta was ultracentrifuged at 28,500 rpm

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(144000×g, 10 °C) for 60 min (Beckman L8-80M ultracentrifuge, SW 40 Ti rotor), and the

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aqueous phase (in the middle between solid and oil) was filtered through a 0.22 μm

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filter. The filtrate containing the micellar phase was extracted and dried under a

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nitrogen stream, redissolved in 0.5 mL n-hexane, and quantitated by HPLC as described

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previously. Bioaccessibility was calculated by the following formula:

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Cytotoxicity of BC micelles

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Cell viability, a commonly used indicator of in vitro cytotoxicity, was evaluated by the

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Prior to

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analyzing the uptake of BC micelles from in vitro digestion, the potential cytotoxicity of

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obtained BC aqueous fraction was evaluated according to previously published

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method.29 Caco-2 cells were incubated in DMEM containing 10% FBS, 1× nonessential

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amino acid (NAA), and 1× penicillin and streptomycin. Caco-2 cells were seeded at a

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density of 1.2×105 cells/well of 96-well plates and incubated (Sanyo, Osaka, Japan) at 37

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˚C in 90% humidity and 5% CO2. After 48 h incubation, the media were discharged, and

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BC micelles after digestion diluted with DMEM at different ratio (0-100 times) were

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added to the wells. After 4 h incubation at 37 ˚C the nano-particles containing media

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were replaced with 100 μL of DMEM containing MTT (10 μL, 5 mg/mL in 1× PBS, pH 7.2)

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and incubated for 2 h at 37 ˚C. Finally, the supernatant was removed and 100 μL of

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dimethyl sulfoxide (DMSO) added. The 96-well plates were analyzed with a Multilabel

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microplate counter (Victor3, PerkinElmer, Waltham, MA) at 570 nm. Cell viability was

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calculated by the percentage of absorbance relative to control (treated with DMEM only).

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Cellular uptake

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Caco-2 cells were incubated in DMEM supplemented 10% FBS, 1× nonessential amino

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acid (NAA), and 1× penicillin and streptomycin at 37 ˚C under 90% humidity and with 5%

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CO2 in an incubator (Sanyo, Osaka, Japan) according to previously described method.30

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Caco-2 cells were seeded at a density of 120,000 cells/ 25 cm2 flask. The medium was

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changed every other day. After five days, the cell monolayers were observed with a

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optical microscope (Leica, IL, U.S.) to ensure that the confluence reached approximately

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90%. Caco-2 cell monolayers were washed with DMEM three times. Following 4 h

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incubation at 37 ˚C in a CO2 incubator, the supernatants were removed and cell

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monolayers were washed three times with pre-cooled PBS solution to stop uptake and

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clean the surface of cell monolayers. After that, 1.0 mL 10% ethanol PBS solution was

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added to dissociate cell monolayers. Cell suspensions were obtained with cell scrapers.

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All of the samples were stored at -80 °C before BC analysis in less than one week.

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Protein content analysis

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Caco-2 cell protein was determined using bicinchoninic acid (BCA) assay at 560 nm with bovine serum albumin as the standard.31

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β-carotene extraction and analysis

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BC was extracted from nanoemulsions and micelles using n-hexane and ethanol32 and

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quantitated by HPLC as described in the section. Extraction was performed by de-

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emulsifying 0.2 mL of sample in a 10 mL Pyrex® glass tube with screw cap by adding 1.0

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mL ethanol, followed 1.5 mL n-hexane immediately, and the contents vortexed for 10

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seconds. The solvent layers were allowed to separate for 1-2 minutes, and the upper

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yellow colored supernatant was transferred to a 10 mL volumetric flask. The extraction

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was repeated with 1.5 mL n-hexane three times until the ground layer was clear. The

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extracts were brought up to 10 mL volume. Each sample was analyzed in triplicate.

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25 μL of 5 μg/mL trans-beta-Apo-8’-carotenal in ethanol was added to 0.4 mL Caco-2

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cell suspension as an internal standard. The cell suspensions were extracted three times

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with ethanol:n-hexane (1:2, v:v). Organic fractions were combined and the extract

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concentrated under a stream of nitrogen gas at 40 ˚C. The BC extract was dissolved in

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0.1 mL of methanol:dichloromethane (1:1, v:v) containing 0.1 % BHT for HPLC analysis.

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The recovery of trans-beta-Apo-8’-carotenal from Caco-2 cells exceeded 94 %.

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High-Performance Liquid Chromatography (HPLC)

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BC extract from nanoemulsions, micelles and Caco-2 cells was quantified using an

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Agilent 1100 HPLC system with DAD UV-vis absorption detector (Agilent, Santa Clara, CA)

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as previously described.10, 30 A C30 reverse-phase analytical column (YMC Carotenoid,

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250×4.6 mm i.d., 5μm, YMC, Inc., Wilmington, NC) was used to separate the carotenoids

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preceded by a reverse-phase C18 guard column (50×3.0 mm i.d., 5μm, YMC, Inc.,

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Wilmington, NC) with a flow rate of 1 mL min-1 at room temperature. The injection

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volume was 20 μL and the detection wavelength was 450 nm. The chromatography

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conditions were as follows: Solvent A: methanol: acetonitrile: H2O (84:14:2, v/v/v),

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solvent B: dichloromethane. The solvent gradient program was: 80A:20B to 45A:55B

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from 0-15 min, and 45A:55B for 15-20 min, and 80A:20B from 20-25 min. The standard

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curve of the absorption peak area versus BC concentration was plotted and fitted with a

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linear function.

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Statistical Analysis

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All experiments were performed at least three times and were reported as mean ±

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standard deviation. The data were analyzed by the analysis of variance (ANOVA) using

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the SPSS 17.0 package (IBM, New York). Duncan’s multiple range test was used to

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determine the significant differences of the mean values (P

Effects of Lipids on in Vitro Release and Cellular Uptake of Î²-Carotene

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