Progress 10/01/09 to 09/30/13
Outputs
Target Audience: Scientifc community and food industries Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Through scientific publication and scientific conferences and workshops. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Summary: Polymethoxyflavones (PMFs) are bioactive flavonoids found in citrus fruits that have been shown to have potential health promoting properties. However, their application as nutraceuticals in functional foods and beverages is currently limited due to their low water solubility and high melting point. The oral bioavailability of lipophilic compounds can be enhanced by promoting their intestinal lymphatic transport through co-administration with digestible lipids. We investigated the effects of chylomicron-mediated intestinal lymphatic transport on the bioavailability of 5-hydroxy-6, 7, 8, 3', 4'-pentamethoxylflavone (5-HPMF), one of representative PMFs in Caco-2 cells. Our results demonstrated that oleic acid and bile acid promoted secretion of chylomicrons in Caco-2 cells, with mean diameter ranged from 70 to 150 nm. The intracellular level of 5-HPMF increased 3-fold by co-incubation with the mixed micelle solution. Moreover, the basolateral level of 5-HPMF increased 3-fold due to enhanced chylomicron-mediated transport. Overall, our results demonstrated for the first time that the bioavailability of polymethoxyflavones can be enhanced by promoting their incorporation into chylomicrons. The Caco-2 cell line is widely accepted as a reliable model for studying the intestinal absorption of lipids and lipoprotein production. We used this model to study the influence of a model fatty acid digestion product (oleic acid) on the absorption and transport of a highly lipophilic bioactive compound (5-HPMF). Oleic acid is a monounsaturated fatty acid that is commonly present in the lipid phase of foods. Our results showed that the presence of mixed micelles containing oleic acid and taurocholic acid (OA-TC) increased the cellular absorption and transport of 5-HPMF (Fig. 3B and Fig. 3C). We also showed that the presence of OA-TC stimulated the production of lipoproteins, such as chylomicrons and VLDL (Fig. 4), which would account for the observed effects. Results and discussion Bioactive lipophilic molecules that are incorporated into chylomicrons are transported into the systemic circulation via the lymphatic system, rather than the portal vein, thereby bypassing first-pass metabolism 27. Previous researchers have identified that oleic acid is able to stimulate synthesis of both apo-B lipoproteins and triglycerides without damaging the monolayer of Caco-2 cells 11, 28. Since oleic acid is highly hydrophobic, it was necessary to mix it with bile salts (TC) so as to form stable colloidal dispersions that could be applied to the Caco-2 cells. We showed that OA:TC mixed micelle solutions were almost transparent and that they contained relatively small colloidal particles, which were probably vesicles (Fig. 2). Previous studies suggest that TC may also enhance the expression of apo-B by enterocytes 11. All experiments in the present study were therefore performed using a molar ratio of 1.6:0.5 OA-to-TC, since this has previously been shown to stimulate maximum amounts of apo-B production in CM fractions 16. The transepithelial electrical resistance (TEER) is widely used as a means to determine the cell-to-cell integrity of cell culture monolayers. Previous studies showed that the TEER of Caco-2 cell monolayers significantly decreased after being exposed to 5 to 30 mM of oleic acid for 180 minutes 29. However, our results exhibited little influence of oleic acid on TEER for any of the three treatments studied. This may be related to the relatively low concentrations of OA and TC used in the present study. Our results also demonstrated that 5-HPMF did not change the integrity of the Caco-2 cell monolayers when used at a concentration of 5 mM. The presence of TC improved the transport of 5-HPMF across the cells compared to the control (Fig. 3B), which can be attributed to its ability to enhance the permeability of the cell monolayer. Nevertheless, the accumulation of 5-HPMF within the Caco-2 cells was not higher than the control (Fig. 3C). In addition, the amount of 5-HPMF present in the basolateral side noticeably decreased at longer incubation times. This may be associated with the metabolism of 5-HPMF as previously demonstrated in our laboratory30. The presence of oleic acid in the mixed micelles appeared to have a transport-enhancing effect: there was an increase in cellular accumulation relative to the control (Fig. 3C) and an increase in the rate and extent of basolateral 5-HPMF (Fig. 3B). One reason is that OA and TC form mixed micelles (vesicles) that can incorporate 5-HPMF, thus increasing its solubility and transport within the aqueous phase. The formation of chylomicrons by the Caco-2 cells was confirmed using electron microscopy (Fig. 4). Lipoproteins with particle diameters ranging from about 20 to 150 nm were found in the basolateral side of the Caco-2 cells (Fig. 5), with about 30% of them being chylomicrons and 70% VLDL particles. The presence of these lipoproteins would account for the observed increase in 5-HPMF transport across the cells. Previous studies have also illustrated that different types of fatty acids affect the number and size of the chylomicrons formed, such as the saturation extent and the chain length 31, 32. In addition, encapsulation of 5-HPMF within lipid nanoparticles may improve its bioavailability by inhibiting its metabolism within and outside the cells. We intend to carry out further studies in this area in future. Conclusion In summary, this in vitro study showed a 3-fold enhanced transport of polymethoxyflavones across Caco-2 cells in the presence of model mixed micelles. We postulate that the OA-TC mixed micelles (vesicles) solubilize 5-HPMF in the apical medium, which facilitates the intracellular uptake of 5-HPMF by the enterocytes. The presence of oleic acid within the cells stimulates the secretion of chylomicrons that incorporate the lipophilic 5-HPMF molecules. Chylomicron formation may increase the bioavailability of lipophilic compounds such as 5-HPMF in a number of ways: (i) increasing the amount that is transported across the cells; (ii) protecting them from metabolism within the cells; (iii) preventing first-pass metabolism in the liver. This research provides a strong base for further studies on the design of colloidal delivery systems that can increase the bioavailability of lipophilic compounds in foods.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yao, M., Chen, J., Zheng, J., Song, M., McClements, D.J., Xiao, H. Enhanced lymphatic transport of bioactive lipids: Cell culture study of polymethoxyflavone incorporation into chylomicrons. Food & Funct., 2013, DOI: 10.1039/C3FO60335K
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Oil phase preparation: PMF powder (1 wt%) was added into a heated carrier oil (∼90 degree C) and the system was continuously stirred until the PMF was completely dissolved. Three kinds of carrier oil were used in this study: corn oil; MCT; and, orange oil. Aqueous phase preparation: Emulsifier solutions were prepared by dispersing 1.0 wt% emulsifier (BLG, Tween 20, Lyso-Lecithin, or DTAB) into 5 mM phosphate buffer solution and stirring for at least 2 h to ensure complete dissolution. The protein solutions were kept overnight at 4 degree C to ensure complete hydration. All emulsifier solutions were warmed prior to use to avoid PMF crystallization when the oil and aqueous phases were brought into contact. Emulsion preparation: A stock emulsion was prepared by homogenizing 5 wt% oil phase (0 or 1 wt% PMF in carrier oil) with 95 wt% aqueous phase (1 wt% emulsifier in buffer solution, pH 7.0) with a high-speed blender for 2 min (M133/1281-0, Biospec Products, Inc., ESGC, Switzerland) followed by five passes at 12K psi through a high pressure homogenizer (Microfluidics M-110Y, F20Y 75 microm interaction chamber, Newton, MA). The oil and aqueous phases were kept warm (>50 degree C) during mixing and homogenization, otherwise the PMF would crystallize and form a gel in the oil phase Particle size and zeta-potential measurements: The particle size and zeta-potential of the particles in the emulsions were determined using a commercial dynamic light scattering and micro-electrophoresis device (Nano-ZS, Malvern Instruments, Worcestershire, UK). The samples were diluted 100 times in buffer solution (5 mM phosphate buffer, pH 7) at room temperature before measurement. The particle size data are reported as the intensity-weighted mean particle diameter, while the particle charge data are reported as the zeta-potential (Malvern Instruments, Worcestershire, UK). The saturation concentrations of PMF in oil (CO,Sat) and water (CW,Sat) phases were measured by spectrophotometry Oil phases: A weighed amount of PMF powder was added to 10 mL oil and then stirred for 1 h at 90-100 degree C to fully dissolve the PMF crystals, which resulted in a clear yellow PMF/oil solution. These solutions were then cooled to 25 degree C and stored for 72 h, which led to crystal formation in samples containing sufficiently high PMF levels. After 72 h storage, the samples were centrifuged (3500 rpm, 15 min) and then 10 microl of supernatant was collected and mixed with 9.99 mL of chloroform. The absorbance was then measured at 327 nm using a UV visible spectrophotometer and the PMF concentration was determined using a previously established calibration curve Water phases: Supersaturated PMF solutions were created by dissolving PMF powder into DMSO. The PMF-DMSO solution was then added to a phosphate buffer solution and mixed thoroughly for 5 s using a Vortex, which led to the formation of crystals. The sample was then centrifuged to remove any non-dissolved PMF. The supernatant was collected and the PMF concentration was determined using a UV-visible spectrophotometer and a previously established calibration curve. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Polymethoxyflavones (PMFs) extracted from citrus peel exhibit potent anti-cancer activity, but are highly hydrophobic molecules with poor solubility in both water and oil at ambient and body temperature, which limits their bioavailability. The possibility of encapsulating PMFs within nanoemulsion-based delivery systems to facilitate their application in nutraceutical and pharmaceutical products was investigated. The influence of oil type (corn oil, MCT, orange oil), emulsifier type (beta-lactoglobulin, lyso-lecithin, Tween, and DTAB), and neutral cosolvents (glycerol and ethanol) on the formation and stability of PMF-loaded nanoemulsions was examined. Nanoemulsions (r<100 nm) could be formed using high pressure homogenization for all emulsifier types, except DTAB. Lipid droplet charge could be altered from highly cationic (DTAB), to near neutral (Tween), to highly anionic (beta-lactoglobulin, lyso-lecithin) by varying emulsifier type. PMF crystals formed in all nanoemulsions after preparation, which had a tendency to sediment during storage. The size, morphology, and aggregation of PMF crystals depended on preparation method, emulsifier type, oil type, and cosolvent addition. These results have important implications for the development of delivery systems for bioactive components that have poor oil and water solubility at application temperatures.
Publications
- Food Hydrocoll. 2012 Jun 1;27(2):517-528. Epub 2011 Sep 17.Nanoemulsion-based delivery systems for poorly water-soluble bioactive compounds: Influence of formulation parameters on Polymethoxyflavone crystallization. Li Y, Zheng J, Xiao H, McClements DJ. 2012.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: We conducted the following experiments: 1. Nanoemulsion and Conventional Emulsion Preparation and Characterization Oil-in-water conventional emulsions or nanoemulsions containing curcumin were prepared. An aqueous emulsifier solution containing 1 wt% protein was prepared by dispersing β-lactoglobulin into PBS and stirring for at least two hours to ensure complete hydration. The oil phase was prepared by adding 0.15 wt% curcumin into the heated oil (60 C), magnetically stirring for 10 min, and then sonicating for 20 min. Coarse emulsions were prepared by homogenizing 10 wt% oil phase with 90 wt% aqueous emulsifier solution using a M133/1281 high-speed blender (Biospec Products, Inc., ESGC, Switzerland) for 2 min at room temperature. Nanoemulsions were prepared by passing the coarse emulsions through a high pressure homogenizer five times at 9000 psi (~620 bars) (Microfluidics M-110Y Microfluidize, MFIC Corporation, Newton, MA, USA) 2.In Vitro Digestion The dynamic in vitro digestion model used was a modification of those described previously. Emulsion and buffer solution (30.0 mL in total) were placed in a clean beaker in a water bath at 37.0 C for 10 min and adjusted to pH 7 with NaOH solution (range from 0.05 - 1M). Then, 4.0 mL of bile extract solution containing 187.5 mg of bile extract (pH 7.0, PBS) and 1.0 mL of CaCl2 solution containing 110 mg of CaCl2 (pH 7.0, PBS) was added into the 30 mL emulsion under stirring. The resultant mixture was then adjusted to pH 7. Finally, 2.5 mL of freshly prepared pancreatin suspension containing 60 mg of lipase (pH 7.0, PBS) was added to the mixture. The bile solution and lipase solution concentrations were 5.0 mg/mL and 1.6 mg/mL respectively. At this point, an automatic titration experiment was started. The pH-stat (Metrohm, USA Inc.) was used to monitor and control the pH (at pH 7) of the digestion solution. The volume of added NaOH solution (500 mM) was assumed to be equal to the amount of free fatty acids generated by lipolysis of the initial triacylglycerols. 3. Bioaccessibility Determination After in vitro digestion, emulsions were centrifuged (12,500 rpm, Sorvall RC 6C Plus, DuPont) at 25 C for 30 min. The emulsions separated into an opaque sediment phase at the bottom, a clear micelle phase in the middle, and sometimes an oily or creamed phase at the top. Aliquots (5 mL) of the micelle phase were collected using a syringe, vortexed with 5 mL of chloroform, and then centrifuged at 1,750 rpm at room temperature for 10 min (Fisher Scientific 225A, Fisher). The bottom chloroform layer was collected, while the top layer was vortexed with another 5 mL of chloroform, and centrifuged (1750 rpm) at room temperature for 10 min. The second bottom chloroform layer was added to the previously set aside chloroform layer, mixed, and analyzed by UV-VIS spectrophotometer (Ultrospec 3000 Pro, GE) with chloroform used as the blank. PARTICIPANTS: Kashif Amhed, Yan Li, Hang Xiao. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Curcumin has been reported to have a variety of biological activities, including anti-oxidant, anti-inflammatory, and anti-carcinogenic properties. However, its application as a functional ingredient is currently limited because of its poor water-solubility and bioaccessibility. Our study investigated the impact of different lipid-based formulations on the encapsulation and bioaccessibility of curcumin. Oil-in-water nanoemulsions (r < 100 nm) or conventional emulsions (r > 100 nm) were prepared with different lipid phases: long, medium, and short chain triacylglycerols (LCT, MCT and SCT, respectively). The maximum amount of curcumin that could be loaded into the lipid phase decreased as the molecular weight of the lipid phase increased: SCT > MCT > LCT. An in vitro model simulating the small intestine digestion conditions was used to characterize the rate and extent of lipid phase digestion. A centrifugation method was used to determine the bioaccessibility of curcumin, i.e., the fraction of curcumin released into mixed micelles after digestion. The rate and extent of lipid digestion depended on triacylglycerol type: initial digestion rate, SCT > MCT > LCT; final extent of digestion, MCT > SCT > LCT. The bioaccessibility of curcumin decreased in the following order MCT > LCT >> SCT. The bioaccessibility of curcumin appeared to be slightly higher in conventional emulsions than in nanoemulsions. These results have important implications for designing delivery systems to encapsulate and release highly lipophilic functional ingredients.
Publications
- 1. Kashif Ahmed, Yan Li, David Julian McClements, and Hang Xiao, (2011) Nanoemulsion- and Emulsion-based Delivery Systems for Curcumin: Encapsulation and Release Properties. Food Chemistry DOI: 10.1016/j.foodchem.2011.11.039.
- 2. Cheng Qian, Eric Decker, Hang Xiao, and David Julian McClements (2011) Solid Lipid Nanoparticles: Effect of Carrier Oil and Emulsifier Type on Phase Behavior and Physical Stability, J Am Oil Chem Soc., DOI 10.1007/s11746-011-1882-0.
- 3. Cheng Qian, Eric Decker, Hang Xiao, and David Julian McClements (2011) Comparison of Biopolymer Emulsifier Performance in Formation and Stabilization of Orange Oil-in-Water Emulsions. J. Am. Oil Chem. Soc. 88: 47-55.
- 4. Yan Li, Min Hu, Yumin Du, Hang Xiao, David Julian McClements. (2011) Control of Lipase Digestibility of Emulsified Lipids by Encapsulation within Calcium Alginate Beads, Food Hydrocolloids, 25 (1): 122-130.
- 5. Yan Li, Min Hu, Hang Xiao, Yumin Du, Eric Andrew Decker and David Julian McClements (2010) Controlling the functional performance of emulsion-based delivery systems using multi-component biopolymer coatings, Eur J Pharm Biopharm. 76 (1): 38-47.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: We have conducted following experiments: Emulsion Preparation Emulsion Preparation for Ostwald Ripening Studies. Lipid phases were prepared with different tributyrin-to-corn oil mass ratios (from 0 to 100% corn oil). Aqueous phases were prepared by dispersing 0.5 wt% emulsifier in buffer (5 mM phosphate, pH 7.0). Oil-in-water emulsions were then prepared that consisted of 5% lipid phase and 95% aqueous phase. Coarse emulsions were prepared by homogenizing 5% lipid phase and 95% aqueous phase in a high-speed blender for 2 min at room temperature. Fine emulsions were then prepared by passing the coarse emulsions through a high pressure homogenizer. The emulsions were passed through the homogenizer 5 times at 612 bar at ambient temperature. After preparation the emulsions were stored at 25 degree Celsius. Emulsion Preparation for Cancer Cell Studies. Oil-in-water emulsions were prepared with 20% lipid phase and 80% aqueous phase. The lipid phase consisted of pure corn oil (control) or 50% corn oil and 50% tributyrin. The aqueous phase consisted of emulsifier dissolved in de-ionized water. The remainder of the emulsion preparation protocol was the same as that used for the Ostwald ripening experiments described above. Emulsion Characterization Particle size measurement. The mean particle sizes (z-average) of the emulsions were measured using dynamic light scattering, with each individual measurement being the average of 13 runs. The emulsions were diluted prior to analysis using either distilled de-ionized water (cell culture experiments) or buffer solution to avoid the effects of multiple scattering. The aqueous phases used to dilute the emulsions were saturated with tributyrin, to avoid migration of tributyrin from the droplets to the surrounding water upon emulsion dilution. Results are reported as the average of 3 measurements on freshly prepared samples. Cell Culture Experiments Cell cultures. The HT29 human colon carcinoma cells were maintained in McCoy 5A media supplemented with 5% heat inactivated FSB, and 100 units/mL of penicillin, and 0.1 mg/mL of streptomycin at 37 degree Celsius with 5% CO2 and 95% air. Cells were kept sub-confluent and media were changed twice a week. All cells used were between 3-30 passages. Treatment procedures. HT29 (7500 cells/well) cells were seeded in 96-well plates. After 24h, cells were treated with serial concentrations of different emulsions in 0.2 mL of serum complete media. After 48h, cells were subject to 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltertrazolium bromide (MTT) assay. MTT assay. Cells proliferation was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltertrazolium bromide (MTT) assay. Media were replaced by 0.1 mL fresh media containing 0.5mg/mL of MTT. After 2 hours of incubation at 37 degree Celsius, MTT-containing media were removed and the reduced formazan dye in the cells was solubilized by adding 0.1 mL DMSO to each well. After gentle mixing, the absorbance was monitored at 550 nm using a 96-well plate reader (Biotek). PARTICIPANTS: Hang Xiao: Principal Investigator; David Julian McClements: Co-PI; Eric Decker: Co-PI; Yan Li: Graduate student. TARGET AUDIENCES: Scientific community; Public; Food policy maker; Food industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Tributyrin, a short-chain triglyceride oil used as a food additive, has been reported to be a potential preventive agent against colon cancer. The purpose of this study was to develop tributyrin delivery systems based on food-grade oil-in-water emulsions that could potentially be incorporated into foods. Emulsions containing only tributyrin as the lipid phase were highly unstable to droplet growth through an Ostwald ripening (OR) mechanism because of the relatively high water-solubility of this low molecular weight triacylglycerol. The stability of the emulsions to OR could be greatly improved by incorporating greater than 15-25% corn oil (a food-grade oil with a low water-solubility) into the lipid phase. In addition, the tendency for droplet sedimentation to occur was reduced because the density contrast between the lipid and water phases was reduced in the mixed tributyrin/corn oil systems. The potential anti-carcinogenic ability of the tributyrin emulsions was demonstrated using a cell culture model. Treatments with emulsions containing tributyrin significantly inhibited the viability of HT29 colon carcinoma cells. These results have important implications for the development and testing of nutraceuticals encapsulated in food-grade delivery systems as anti-cancer agents.
Publications
- Emulsion-based delivery systems for tributyrin, a potential colon cancer preventative agent. Li Y, Le Maux S, Xiao H, McClements DJ. J Agric Food Chem. 2009, 57(19):9243-9.
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