Article pubs.acs.org/JAFC
Lecithin-Based Nano-emulsification Improves the Bioavailability of Conjugated Linoleic Acid Wan Heo,∥ Jun Ho Kim,∥ Jeong Hoon Pan, and Young Jun Kim* Department of Food and Biotechnology, Korea University, Sejong 30019, Republic of Korea S Supporting Information *
ABSTRACT: In this study, we investigated the effects of lecithin-based nano-emulsification on the heat stability and bioavailability of conjugated linoleic acid (CLA) in different free fatty acid (FFA) and triglyceride (TG) forms. CLA nanoemulsion in TG form exhibited a small droplet size (70−120 nm) compared to CLA nano-emulsion in FFA form (230−260 nm). Nano-emulsification protected CLA isomers in TG form, but not in free form, against thermal decomposition during the heat treatment. The in vitro bioavailability test using monolayers of Caco-2 human intestinal cells showed that nanoemulsification increased the cellular uptake of CLA in both FFA and TG forms. More importantly, a rat feeding study showed that CLA content in small intestinal tissues or plasma was higher when CLA was emulsified, indicating an enhanced oral bioavailability of CLA by nano-emulsification. These results provide important information for development of nano-emulsionbased delivery systems that improve thermal stability and bioavailability of CLA. KEYWORDS: conjugated linoleic acid, nano-emulsificaion, bioavailability, soybean lecithin, heat stability
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INTRODUCTION Conjugated linoleic acid (CLA) comprises a family of positional and geometric isomers of linoleic acid (LA) and is mainly found in foods such as ruminant meat, dairy products, and milk.1,2 CLA has drawn significant scientific attention in the past few decades due to its beneficial health implications including antioxidative, anticarcinogenic, antiatherosclerotic, antiadipogenic, and immune modulatory activities.3−5 CLA has been approved as generally recognized as safe (GRAS) in a mixture of 60−90% of the cis-9,trans-11, and trans-10,cis-12 isomers in the United States since 2008; thus, it is expected that the consumption of CLA as a dietary supplement or food will increase.6 However, application of CLA as a bioactive ingredient in the food industry has been limited due to its water insolubility and oxidative instability.7 Moreover, it has been suggested that CLA may induce upset stomach, nausea, and loose stools because of its suboptimal absorption rate in the human body.8 Thus, many investigators have tried to improve the oxidative stability and ruminal protection of CLA by physicochemical modifications,7 but there is limited information regarding the bioavailability of CLA in different forms. Nano-emulsification leads to the formation of a nonequilibrium colloidal system where the oil phase is dispersed as fine droplets, usually with particle sizes from 20 to 500 nm, throughout the aqueous phase stabilized by surfactants.9 Nanoemulsification is a good vehicle to formulate hydrophobic active molecules and has been widely used to improve bioavailability of several bioactive compounds including lipids.10 Soybean lecithin has been shown to be a good surfactant for nanoemulsification, due to its high affinity to cellular membranes leading to an increased absorption of bioactive compounds.11 Recently, Kim et al.8 reported that lecithin-based nanoemulsification of CLA enhanced the antiobesity effects of CLA in rats, but there was no evidence of improved bioavailability. © XXXX American Chemical Society
Two main methods (high-energy and low-energy) have been used to fabricate nano-emulsions. High-energy emulsification utilizes mechanical devices (e.g., high-pressure homogenizer, microfluidizer, ultrasonicators) to create intensely disruptive forces that break up the oil and water phases to form nanosized droplets.12 This method allows for a greater control of particle size and a large choice of composition, which affect the stability and rheology of the emulsion.13 Therefore, this study was designed to elucidate whether lecithin-based nano-emulsification of CLA produced by a high-pressure homogenizer improves its bioavailability and heat stability in in vitro and in vivo systems. To determine whether esterification of CLA affects its bioavailability, we compared two different forms [free fatty acid (FFA) and triglyceride (TG)] of CLA in this study.
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MATERIALS AND METHODS
Materials. CLA was provided by HK Biotech Co., Ltd. (Jinju, Korea). The composition of the CLA isomers was 38.6% cis-9,trans-11, 43.3% trans-10,cis-12, and 3.5% other CLA isomers. Soybean lecithin and glycerol were purchased from H&A Pharmachem (Bucheon, Korea). Other chemicals used were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Preparation of CLA Nano-emulsions. A schematic diagram for the preparation of CLA nano-emulsions using a high-pressure homogenizer is shown in Figure 1. CLA nano-emulsions in both FFA and TG forms were composed of 20% CLA, 5% soybean lecithin (69.9% phosphatidylcholine and 8.4% phosphatidylethanolamine), and 65% aqueous glycerol solution. Preparation of pre-emulsions was performed as described previously14 with some modifications. Soybean lecithin was dissolved into the water at 70 °C, and the CLA was stirred in the aqueous glycerol solution at 400 rpm and 70 °C for about 10 Received: November 12, 2015 Revised: January 29, 2016 Accepted: January 29, 2016
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DOI: 10.1021/acs.jafc.5b05397 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Schematic diagram for the preparation of CLA nano-emulsions. Pre-emulsion of CLA, lecithin (surfactant), and glycerol (cosurfactant) in water was subjected to high-pressure homogenization. The sample flowed through the microchannel onto an impingement area, resulting in very fine particle of submicromemter range at 35,000 psi. The prepared coarse emulsion was continuously passed into the reaction chamber to obtain the desired particle size (four cycles). Conventional oil-in-water emulsions, with CLA surrounded by a thin interfacial layer consisting of lecithin and glycerol, were used in this study. min. The mixture was emulsified by stirrer at 1000 rpm at 70 °C for 20 min. Subsequently, the pre-emulsion obtained was passed through a high-pressure homogenizer (Micronox, Seongnam, Korea) at a pressure of 35,000 psi for four cycles. The obtained samples were cooled to room temperature and stored at 4 °C. Determination of Particle Size and Zeta-Potential. The droplet size and zeta-potential of CLA nano-emulsions were determined by photon correlation spectroscopy (Malvern Nano ZS90, Malvern, UK). The samples (1 mL) were dispersed in 100 mL of water and gently mixed by inversion. Dynamic light scattering (DLS) measurements were carried out at 25 °C and a 90° scattering angle. Electrophoresis mobility was measured and zeta-potential was calculated by the Dispersion Technology Software provided by Malvern according to the Henry equation (UE = 2ezf(Ka)/3h). Cryogenic Transmission Electron Microscopy. All samples were diluted approximately 1000-fold with distilled water. For cryogenic transmission electron microscopy (cryo-TEM), 3 μL samples were applied on a perforated lacey carbon film grid (200 mesh copper; Ted Pella, Redding, CA, USA) and blotted with filter paper (Whatman, 1 μm) for approximately 3 s. After blotting, the grid was immediately plunged into precooled liquid ethane for flash freezing. The cryo-grid was held in a Gatan 655 cryo-holder (Gatan, USA) and transferred into TEM (JEOL JEM-2010 with 200 kV LaB6 filament) at −185 °C. Samples were observed under minimal dose conditions at −185 °C. Micrographs were recorded by a Gatan 832 charge-coupled device camera at a magnification of 10,000−50,000× and at a defocus of 3−5.46 μm. Fatty Acid Analyses. All of the chemicals used for gas chromatography analysis were of analytical grade and purchased from Sigma (St. Louis, MO, USA). One milliliter of plasma or 100 mg of tissue sample was used to extract lipids with 5 mL of hexane/ isopropanol (3:2 vol/vol). Fatty acid methyl esters were prepared by reaction with 4% HCl in methanol for 20 min at 60 °C15 and identified by using an Agilent Technologies 7890A gas chromatograph with a flame ionization detector (Agilent Technologies, Palo Alto, CA, USA). Fatty acid methyl esters were separated using a Supelcowax-10 fused silica capillary column (100 m × 0.32 mm i.d., 0.25 μm film thickness; Supelco, Inc., Bellefonte, PA, USA) with a 1.2 mL/min of helium flow. The gas chromatograph was operated at a temperature of 140 °C for 5 min, followed by heating at 2 °C/min to 240 °C, and holding for 30 min. Both the injector and detector were maintained at 260 °C. One microliter of sample was injected into the column in the split mode (50:1). Heptadecanoic acid (C17:0) was included as an internal reference before the extraction of lipids to determine the recovery of the fatty acids in each sample. The recovery of methylated fatty acids calculated in a comparison to the internal standard was >80%.
Heat Stability Test. Ten grams of CLA-FFA (FCLA), CLA-TG (TCLA), FCLA nano-emulsion (nano-FCLA) or TCLA nanoemulsion (nano-TCLA) was placed in 20 mL vials and held in an oven at 95 ± 1 °C for 48 h. Immediately after each storage period, changes of the main CLA isomer (cis-9,trans-11, and trans-10,cis-12) contents of oil samples were analyzed by gas chromatography. Values are expressed as a percentage of baseline. The area under the curve (AUC) was calculated for each group during the test. Assessment of CLA Absorption by Caco-2 Human Intestinal Cells. Caco-2 human intestinal cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids, 1% N-(2-hydroxyethyl)piperazine-N′-(2ethanesulfonic acid) (HEPES) buffer (1 M), and 1% penicillin/ streptomycin solution in an atmosphere of 5% CO2 and 95% relative humidity. Cells were seeded at a density of 6 × 104 in six-well plates with transwell inserts (24 mm i.d., 0.4 μm pore size, polycarbonate filter, Corning Costar Co., Cambridge, MA, USA) coated with a collagen layer. Cells were grown and differentiated to confluent monolayers for 21 days. The integrity of the monolayer was monitored by measuring transepithelial electrical resistance (TEER) with a Millicell electrical resistance system (Micropolimetro Millicell-ERS, Millipore Iberia, Spain). During differentiation of Caco-2 cells (15−21 days after seeding), the TEER value was monitored every 48 h. Monolayers with TEER values of >400 Ω cm−2 were used for the transport study.16 Transport experiments were initiated by washing the monolayers with Hank’s balanced salt solution (HBSS) before the stock solutions of samples were added to the apical compartment of the cell. Stock solutions of CLA were prepared in dimethyl sulfoxide (DMSO) and were diluted in medium at a maximum of 0.2% (v/v) DMSO. After 2 h of incubation, the samples were collected from the apical and basolateral compartment and stored at −70 °C until analyzed. In Vivo Bioavailability. Female 10-week-old Sprague−Dawley rats were purchased from Samtako Co., Ltd. (Osan, Korea) and were housed in standard cages under controlled temperature (22 ± 0.5 °C), humidity (50%), and light (light from 9:00 a.m. to 9:00 p.m.) conditions. After 1 week of adaptation, rats were fasted for 6 h, and 1 mL of CLA samples was orally administered five times at 30 min intervals.17 Lecithin and glycerol were added to FCLA and TCLA to replace an equivalent amount of CLA nano-emulsions. Thirty minutes after the final administration, the rats were sacrificed by CO2 gas asphyxiation, and blood samples were collected via cardiac puncture. The small intestine was washed with PBS, and duodenum, jejunum, and ileum were collected and stored at −70 °C until fatty acid analyses. All animal work was completed in compliance with the guidelines of Korea University (Seoul, Korea) for the Ethical Treatment of Laboratory Animals. B
DOI: 10.1021/acs.jafc.5b05397 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 2. Physicochemical properties of CLA nano-emulsions: (A) droplet size of CLA nano-emulsions and void without CLA measured for five cycles of high-pressure homogenization; (B) representative cryo-TEM image of nano-FCLA and nano-TCLA; (C) polydispersity index and (D) zeta-potential determined by photon correlation spectroscopy. Nano-FCLA, CLA-FFA nano-emulsion; nano-TCLA, CLA-TG nano-emulsion. Values represent means ± SE (n = 5). Statistical Analysis. Data were analyzed by one-way ANOVA using SAS software for Windows release 9.2 (SAS Institute Inc., Cary, NC, USA) on the W32_VSHOME platform. The Least Squares Means option using a Tukey−Kramer adjustment was used for multiple comparisons among the treatment groups. Data are shown as the mean ± SE. P values
